U.S. patent number 6,610,702 [Application Number 09/920,140] was granted by the patent office on 2003-08-26 for ammonium salts of inositol hexaphosphate, and uses thereof.
This patent grant is currently assigned to GMP Oxycell, Inc.. Invention is credited to Jean-Marie Lehn, Yves Claude Nicolau, Stephane P. Vincent.
United States Patent |
6,610,702 |
Lehn , et al. |
August 26, 2003 |
Ammonium salts of inositol hexaphosphate, and uses thereof
Abstract
The present invention comprises compounds, compositions thereof,
and methods capable of delivering inositol hexaphospahte (IHP) to
the cytoplasm of mammalian cells. In certain embodiments, the
present invention relates to compounds, compositions thereof, and
methods that enhance the ability of mammalian red blood cells to
deliver oxygen, by delivering IHP to the cytoplasm of the red blood
cells.
Inventors: |
Lehn; Jean-Marie (Strasbourg,
FR), Nicolau; Yves Claude (Newton, MA), Vincent;
Stephane P. (Strasbourg, FR) |
Assignee: |
GMP Oxycell, Inc. (Fort
Lauderdale, FL)
|
Family
ID: |
22830769 |
Appl.
No.: |
09/920,140 |
Filed: |
August 1, 2001 |
Current U.S.
Class: |
514/305; 514/311;
514/415; 546/133; 546/164; 548/494 |
Current CPC
Class: |
C07C
211/06 (20130101); C07C 211/07 (20130101); C07C
211/18 (20130101); C07C 211/27 (20130101); C07C
211/35 (20130101); C07C 217/84 (20130101); C07C
229/08 (20130101); C07C 229/36 (20130101); C07D
207/16 (20130101); C07D 209/20 (20130101); C07D
211/12 (20130101); C07D 215/06 (20130101); C07D
257/02 (20130101); C07D 295/033 (20130101); C07D
295/13 (20130101); C07D 453/02 (20130101); C07F
9/117 (20130101); C07C 2601/08 (20170501); C07C
2601/14 (20170501); C07C 2601/18 (20170501); C07C
2602/08 (20170501); C07C 2602/42 (20170501); C07C
2603/74 (20170501) |
Current International
Class: |
C07C
229/00 (20060101); C07C 211/35 (20060101); C07C
229/36 (20060101); C07C 229/08 (20060101); C07C
211/06 (20060101); C07C 211/07 (20060101); C07C
211/27 (20060101); C07C 217/84 (20060101); C07C
211/18 (20060101); C07C 217/00 (20060101); C07C
211/00 (20060101); C07D 209/00 (20060101); C07D
207/00 (20060101); C07D 207/16 (20060101); C07D
257/00 (20060101); C07D 211/00 (20060101); C07D
209/20 (20060101); C07D 211/12 (20060101); C07D
257/02 (20060101); C07D 453/00 (20060101); C07D
215/00 (20060101); C07D 215/06 (20060101); C07D
295/027 (20060101); C07D 453/02 (20060101); C07D
295/13 (20060101); C07F 9/00 (20060101); C07D
295/00 (20060101); C07D 295/033 (20060101); C07F
9/117 (20060101); C07D 453/02 (); C07D 211/12 ();
C07D 215/06 (); C07D 209/20 (); A61K 031/661 () |
Field of
Search: |
;546/133,164 ;548/494
;514/305,311,415 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 146 338 |
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Jun 1985 |
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EP |
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51-108020 |
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Sep 1976 |
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JP |
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55-147295 |
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Nov 1980 |
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JP |
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WO 92/20368 |
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Nov 1992 |
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WO |
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WO 92/20369 |
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Nov 1992 |
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WO |
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WO 93/16688 |
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Sep 1993 |
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WO |
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WO 94/21117 |
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Sep 1994 |
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WO |
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WO 95/03068 |
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Feb 1995 |
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WO |
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WO97/42819 |
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Nov 1997 |
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WO |
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WO 01/13933 |
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Mar 2001 |
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WO |
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WO 01/24830 |
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Apr 2001 |
|
WO |
|
Other References
Hirst et al.; "The Modification of Hemoglobin Affinity For Oxygen
and Tumor Radiosensivity by Antilipidemic Drugs", Radiation
Research 112: 164-172, (1987). .
Ogata and McConnell.; "Triphosphate Spin-Label Studies of
Allosteric Interactions In Hemoglobin", Annals of the New York
Academy of Sciences, 222: 56-67, (Dec. 31, 1973). .
Ruckpaul et al.; "Interaction of Hemoglobin with Ions Allosteric
Effects of the Binding of Anions", Biochimica et Biophysica Acta
236:211-221, (1971). .
Benesch and Benesch; "The Effect of Organic Phosphates From the
Human Erythrocyte on the Allosteric Prosperities of Hemoglobin",
Biochemical and Biophysical Research Communications, 26 (2):
163-167, (1967). .
Lalezari et al.; "New Effectors Of Human Hemoglobin: Structure and
Function", Biochemistry 29: 1515-1523, (1990). .
Abraham et al.; "Design, Synthesis, and Testing of Potential
Antisickling Agents. 1. Halogenated Benzyloxy and Phenoxy acids",
J. Med. Chem. 25: 1015-1017, (1982). .
Teisseire et al.; "Physiological Effects of High -P.sub.50
Erythrocyte Trasnfusion on Piglets", Journal of Applied Physiology,
58(4): 1810-1817, (Apr. 1985). .
Brooksbank and Balazs; Superoxide Dismutase and Lipoperoxidation in
Down's Syndrome Fetal Brain, The Lancet 1: 881-882, (Apr. 16,
1983). .
Benesch and Benesch; "Intracellular Organic Phosphates as
Regulators of Oxygen Release by Haemoglobin", Nature, 221: 618-622,
(Feb. 15, 1969). .
Arnone Arthur; "X-ray Diffraction Study of Binding of
2,3-Diphosphoglycerate to Human Deoxyhaemoglobin", Nature 237:
146-149, (May 19, 1972). .
Abraham et al.; "Physiological and X-ray Studies of Potential
Antisickling Agents", Proc. Natl. Acad. Sci. USA, 80:324-328, (Jan.
1983). .
Teisseire et al.; "Long-term Physiological Effects of Enhanced
O.sub.2 Release by Inositol Hexaphosphate-Loaded Erythrocytes",
Proc. Natl. Acad. Sci. USA, 84: 6894-6898, (Oct. 1987). .
Lalezari et al.; "LR16, a Compound with Potent Effects on the
Oxygen affinity of Hemoglobin, on Blood Cholesterol, and on Low
Density Lipoprotein", Proc. Natl. Acad. Sci. USA, 85: 6117-6121,
(Aug. 1988). .
Bruggemann et al.; "Low Oxygen-Affinity Red Cell Produced In a
Large-Volume, Continous-Flow Electroporation System", Tranfusion
35(6): 478-485, (Jun. 1995)..
|
Primary Examiner: Shah; Mukund J.
Assistant Examiner: Liu; Hong
Attorney, Agent or Firm: Gordon; Dana M. Foley Hoag LLP
Parent Case Text
RELATED APPLICATION INFORMATION
This application claims the benefit of priority under 35 U.S.C.
section 119(e) to Provisional Patent Applications Nos. 60/222,089,
filed Aug. 1, 2000. This application is hereby incorporated by
reference in its entirety.
Claims
We claim:
1. A composition represented by structure 1: ##STR22## wherein
nC.sup.+ represents nona-cyclohexylammonium-tri-sodium,
bis-dicyclohexylammonium-deca-sodium, octa-dicyclohexylammonium,
hepta-1-aza-3-hydroxyl-bicyclo[2.2.2]cyclooctanium,
dodeca-1-aza-3-hydroxyl-bicyclo[2.2.2]cyclooctanium,
nona-piperidinium, penta-H.sub.3 N-Phe-OMe, nona-H.sub.3 N-Phe-OMe,
hexa-1-indanylammonium, hepta-2-norbornylammonium,
nona-decahydroquinolinium, hepta-H.sub.3 N-Phe-OEt, hexa-H.sub.3
N-Phe-OEt, octa-H.sub.3 N-sec-Leu-Ot-Bu,
dodeca-diisopropylammonium, octa-H.sub.3 N-Pro-Ot-Bu, deca-H.sub.3
N-Tyr-OEt, tetra-cyclohexyl-1,2-bis-ammonium,
nona-cycloheptylammonium, undeca-cyclopentylammonium, or
undeca-cyclohexylammonium,
penta-(N,N'-dibenzyl)-ethylenediammonium, octa
menthyl-1,8-diammonium, penta cyclohexyl-(1,3-bismethylammonium),
penta (.+-.)-(1,2-trans-diphenyl)-ethylenediammonium, nona
N-cyclohexyl-piperidinium, bis (N.sup.1,N.sup.3
-cyclohexyl)-dipropylenetriammonium, tris
tri-(N-cyclohexyl-2-amino-ethyl)-ammonium, tetra
N,N'-di-(3-(N-cyclohexyl-amino)-propyl)-piperazinium, tris
tri-(N-cycloheptyl-2-amino-ethyl)-ammonium, tri
N,N'-di-(3-(N-cyclooctyl-amino)-propyl)-piperazinium, or bis
N,N',N",N'"-tetrahexyl-cyclam; and A.sup.n- represents a conjugate
base of inositol hexaphosphate, wherein n equals the number of
cations comprised by nC.sup.+.
2. The compound of claim 1, wherein nC.sup.30 represents
nona-cyclohexylammonium-tri-sodium,
bis-dicyclohexylammonium-deca-sodium, octa-dicyclohexylammonium,
hepta-1-aza-3-hydroxyl-bicyclo[2.2.2]cyclooctanium,
dodeca-1-aza-3-hydroxyl-bicyclo[2.2.2]cyclooctanium,
nona-piperidinium, penta-H.sub.3 N-Phe-OMe, nona-H.sub.3 N-Phe-OMe,
hepta-2-norbornylammonium, nona-decahydroquinolinium, hepta-H.sub.3
N-Phe-OEt, hexa-H.sub.3 N-Phe-OEt, dodeca-diisopropylammonium,
octa-H.sub.3 N-Pro-Ot-Bu, deca-H.sub.3 N-Tyr-OEt,
tetra-cyclohexyl-1,2-bis-ammonium, nona-cycloheptylammonium,
undeca-cyclopentylammonium, undeca-cyclohexylammonium, octa
menthyl-1,8-diammonium, penta cyclohexyl-(1,3-bismethylammonium),
nona N-cyclohexyl-piperidinium, bis (N.sup.1,N.sup.3
-cyclohexyl)-dipropylenetriammonium, tris
tri-(N-cyclohexyl-2-amino-ethyl)-ammonium, tetra N,N.sup.'
-di-(3-(N-cyclohexyl-amino)-propyl)-piperazinium, tris
tri-(N-cycloheptyl-2-amino-ethyl)-ammonium, or tris
N,N'-di-(3-(N-cyclooctyl-amino)-propyl)-piperazinium.
3. The compound of claim 1, wherein nC.sup.30 represents
nona-cyclohexylammonium-tri-sodium, octa-dicyclohexylammonium,
nona-decahydroquinolinium, dodeca-diisopropylammonium, octa-H.sub.3
N-Pro-Ot-Bu, nona-cycloheptylammonium, or
undeca-cyclohexylammonium.
4. The compound of claim 1, wherein nC.sup.30 represents
nona-decahydroquinolinium, dodeca-diisopropylammonium, octa-H.sub.3
N-Pro-Ot-Bu, nona-cycloheptylammonium, or
undeca-cyclohexylammonium.
5. The compound of claim 1, wherein nC.sup.30 represents
nona-cycloheptylammonium or undeca-cyclohexylammonium.
6. The compound of claim 1, wherein nC.sup.30 represents
nona-cycloheptylammonium.
7. The compound of claim 1, wherein nC.sup.30 represents
undeca-cyclohexylammonium.
Description
BACKGROUND OF THE INVENTION I. Ischemia
Ischemic insult, i.e., the localized deficiency of oxygen to an
organ or skeletal tissue, is a common and important problem in many
clinical conditions. The problem is especially acute in organ
transplant operations in which a harvested organ is removed from a
body, isolated from a blood source, and thereby deprived of oxygen
and nutrients for an extended period of time. Ischemic insult also
occurs in certain clinical conditions, such as sickle cell anemia
and septic shock, which may result from hypotension or organ
dysfunction. Depending on the duration of the insult, the ischemia
can disturb cellular metabolism and ion gradients, and ultimately
cause irreversible cellular injury and death.
Arguably, heart attacks and stroke are the most widely recognized
example of the damage resulting from ischemia. Myocardial ischemia
is a condition wherein there is insufficient blood supply to the
myocardium (the muscles of the heart) to meet its demand for
oxygen. The ultimate result of persistent myocardial ischemia is
necrosis or death of a portion of cardiac muscle tissue, known as a
myocardial infarct, commonly known as a heart attack.
Insufficient blood supply to the myocardium is generally due to an
obstruction or thrombus in an artery supplying blood to the
myocardium. Another cause can be atrial fibrillation, wherein the
increased heart rate associated with atrial fibrillation increases
the work, and hence the blood demand of the myocardium, while the
atrial fibrillation at the same time reduces the blood supply.
Whereas stroke is defined as a sudden impairment of body functions
caused by a disruption in the supply of blood to the brain. For
instance, a stroke occurs when blood supply to the brain is
interrupted for any reason, including hemorrhage, low blood
pressure, clogging by atherosclerotic plaque, a blood clot, or any
particle. Because of the blockage or rupture, part of the brain
fails to get the supply of blood and oxygen that it requires. Brain
tissue that receives an inadequate supply of blood is said to be
ischemic. Deprived of oxygen and nutrients, nerve cells and other
cell types within the brain begin to fail, creating an infarct (an
area of cell death, or necrosis). As the neurons fail and die, the
part of the body controlled by those neurons can no longer
function. The devastating effects of ischemia are often permanent
because brain tissue has very limited repair capabilities and lost
neurons are typically not regenerated.
Cerebral ischemia may be incomplete (blood flow is reduced but not
entirely cut off), complete (total loss of tissue perfusion),
transient or permanent. If ischemia is incomplete and persists for
no more than ten to fifteen minutes, neural death may not occur.
More prolonged or complete ischemia results in infarction.
Depending on the site and extent of the infarction, mild to severe
neurological disability or death will follow.
To a modest extent, the brain is protected against cerebral
ischemia by compensatory mechanisms, including collateral
circulation (overlapping local blood supplies), and arteriolar
auto-regulation (local smooth muscle control of blood flow in the
smallest arterial channels). If compensatory mechanisms operate
efficiently, slightly diminished cerebral blood flow produces
neither tissue ischemia nor abnormal signs and symptoms. Usually,
such mechanisms must act within minutes to restore blood flow if
permanent infarction damage is to be avoided or reduced. Arteriolar
auto-regulation works by shunting blood from noncritical regions to
infarct zones.
Even in the face of systemic hypotension, auto-regulation may be
sufficient to adjust the circulation and thereby preserve the
vitality and function of brain or heart tissue. Alternatively,
ischemia may be sufficiently prolonged and compensatory mechanisms
sufficiently inadequate that a catastrophic stroke or heart attack
results.
Ischemia is also associated with various clinical conditions, such
as septic shock. Septic shock as a result of hypotension and organ
dysfunction in response to infectious sepsis is a major cause of
death. The manifestations of sepsis include those related to the
systemic response to infection (tachycardia, tachypnea alterations
in temperature and leukocytosis) and those related to organ-system
dysfunction (cardiovascular, respiratory, renal, hepatic and
hematologic abnormalities). Furthermore, the lipopolysaccharide
(LPS) of gram-negative bacteria is considered to be the most
important exogenous mediator of acute inflammatory response to
septic shock. The LPS or endotoxin released from the outer membrane
of gram-negative bacteria results in the release of cytokines and
other cellular mediators, including tumor necrosis factor alpha
(TNF alpha), interleukin-1 (Il-1), interleukin-6 (Il -6) and
thromboxane A2. Extreme levels of these mediators are known to
trigger many pathological events, including fever, shock, and
intravascular coagulation, leading to ischemia and organ failure.
II. Hemoglobin
Hemoglobin is a tetrameric protein which delivers oxygen via an
allosteric mechanism. Oxygen binds to the four hemes of the
hemoglobin molecule. Each heme contains porphyrin and iron in the
ferrous state. The ferrous iron-oxygen bond is readily reversible.
Binding of the first oxygen to a heme releases much greater energy
than binding of the second oxygen molecule, binding of the third
oxygen releases even less energy, and binding of the fourth oxygen
releases the least energy.
In blood, hemoglobin is in equilibrium between two allosteric
structures. In the "T" (for tense) state, hemoglobin is
deoxygenated. In the "R" (for relaxed) state, hemoglobin is
oxygenated. An oxygen equilibrium curve can be scanned to observe
the affinity and degree of cooperativity (allosteric action) of
hemoglobin. In the scan, the Y-axis plots the percent of hemoglobin
oxygenation and the X-axis plots the partial pressure of oxygen in
millimeters of mercury (mm Hg). If a horizontal line is drawn from
the 50% oxygen saturation point to the scanned curve and a vertical
line is drawn from the intersection point of the horizontal line
with the curve to the partial pressure X-axis, a value commonly
known as the P.sub.50 is determined (i.e., this is the pressure in
mm Hg when the scanned hemoglobin sample is 50% saturated with
oxygen). Under physiological conditions (i.e., 37 C, pH=7.4, and
partial carbon dioxide pressure of 40 mm Hg), the P.sub.50 value
for normal adult hemoglobin (HbA) is around 26.5 mm Hg. If a lower
than normal P.sub.50 value is obtained for the hemoglobin being
tested, the scanned curve is considered to be "left-shifted" and
the presence of high oxygen-affinity hemoglobin is indicated.
Conversely, if a higher than normal P.sub.50 value is obtained for
the hemoglobin being tested, the scanned curve is considered to be
"right-shifted", indicating the presence of low oxygen-affinity
hemoglobin.
It has been proposed that influencing the allosteric equilibrium of
hemoglobin is a viable avenue of attack for treating diseases. The
conversion of hemoglobin to a high affinity state is generally
regarded to be beneficial in resolving problems with
(deoxy)hemoglobin-S (i.e., sickle cell anemia). The conversion of
hemoglobin to a low affinity state is believed to have general
utility in a variety of disease states where tissues suffer from
low oxygen tension, such as ischemia and radio sensitization of
tumors. Several synthetic compounds have been identified which have
utility in the allosteric regulation of hemoglobin and other
proteins. For example, several new compounds and methods for
treating sickle cell anemia which involve the allosteric regulation
of hemoglobin are reported in U.S. Pat. No. 4,699,926 to Abraham et
al., U.S. Pat. No. 4,731,381 to Abraham et al., U.S. Pat. No.
4,731,473 to Abraham et al., U.S. Pat. No. 4,751,244 to Abraham et
al., and U.S. Pat. No. 4,887,995 to Abraham et al. Furthermore, in
both Perutz, "Mechanisms of Cooperativity and allosteric Regulation
in Proteins", Quarterly Reviews of Biophysics 22, 2 (1989), pp.
163-164, and Lalezari et al., "LR16, a compound with potent effects
on the oxygen affinity of hemoglobin, on blood cholesterol, and on
low density lipoprotein", Proc. Natl. Acad. Sci, USA 85 (1988), pp.
6117-6121, compounds which are effective allosteric hemoglobin
modifiers are discussed. In addition, Perutz et al. has shown that
a known antihyperlipoproteinemia drug, bezafibrate, is capable of
lowering the affinity of hemoglobin for oxygen (See "Bezafibrate
lowers oxygen affinity of hemoglobin", Lancet 1983, 881).
Human normal adult hemoglobin ("HbA") is a tetrameric protein
containing two alpha chains having 141 amino acid residues each and
two beta chains having 146 amino acid residues each, and also
bearing prosthetic groups known as hemes. The erythrocytes help
maintain hemoglobin in its reduced, functional form. The heme-iron
atom is susceptible to oxidation, but may be reduced again by one
of two systems within the erythrocyte, the cytochrome b5, and
glutathione reduction systems.
Hemoglobin is able to alter its oxygen affinity, thereby increasing
the efficiency of oxygen transport in the body due to its
dependence on 2,3-DPG, an allosteric regulator. 2,3-DPG is present
within erythrocytes at a concentration that facilitates hemoglobin
to release bound oxygen to tissues. Naturally-occurring hemoglobin
includes any hemoglobin identical to hemoglobin naturally existing
within a cell. Naturally-occurring hemoglobin is predominantly
wild-type hemoglobin, but also includes naturally-occurring mutant
hemoglobin. Wild-type hemoglobin is hemoglobin most commonly found
within natural cells. Wild-type human hemoglobin includes
hemoglobin A, the normal adult human hemoglobin having two alpha-
and two beta-globin chains. Mutant hemoglobin has an amino-acid
sequence that differs from the amino-acid sequence of wild-type
hemoglobin as a result of a mutation, such as a substitution,
addition or deletion of at least one amino acid. Adult human mutant
hemoglobin has an amino-acid sequence that differs from the
amino-acid sequence of hemoglobin A. Naturally-occurring mutant
hemoglobin has an amino-acid sequence that has not been modified by
humans. The naturally-occurring hemoglobin of the present invention
is not limited by the methods by which it is produced. Such methods
typically include, for example, erythrocytolysis and purification,
recombinant production, and protein synthesis.
It is known that hemoglobin specifically binds small polyanionic
molecules, especially 2,3-diphosphoglycerate (DPG) and adenosine
triphosphate (ATP), present in the mammalian red cell (Benesch and
Benesch, Nature, 221, p. 618, 1969). This binding site is located
at the centre of the tetrameric structure of hemoglobin (Arnone,
A., Nature, 237, p. 146, 1972). The binding of these polyanionic
molecules is important in regulating the oxygen-binding affinity of
hemoglobin since it allosterically affects the conformation of
hemoglobin leading to a decrease in oxygen affinity (Benesch and
Benesch, Biochem. Biophys. Res. Comm., 26, p. 162, 1967).
Conversely, the binding of oxygen allosterically reduces the
affinity of hemoglobin for the polyanion. (Oxy) hemoglobin
therefore binds DPG and ATP weakly. This is shown, for example, by
studies of spin-labeled ATP binding to oxy- and deoxyhemoglobin as
described by Ogata and McConnell (Ann. N.Y. Acad. Sc., 222, p. 56,
1973). In order to exploit the polyanion-binding specificity of
hemoglobin, or indeed to perform any adjustment of its
oxygen-binding affinity by chemically modifying the polyanion
binding site, it has been necessary in the prior art that
hemoglobin be deoxygenated. However, hemoglobin as it exists in
solutions, or mixtures exposed to air, is in its oxy state, i.e.,
(oxy)hemoglobin. In fact it is difficult to maintain hemoglobin
solutions in the deoxy state, (deoxy)hemoglobin, throughout a
chromatographic procedure. Because of these difficulties, the
technique of affinity chromatography has not been used in the prior
art to purify hemoglobin.
Hemoglobin has also been administered as a pretreatment to patients
receiving chemotherapeutic agents or radiation for the treatment of
tumors (U.S. Pat. No. 5,428,007; WO 92/20368; WO 92/20369), for
prophylaxis or treatment of systemic hypotension or septic shock
induced by internal nitric oxide production (U.S. Pat. No.
5,296,466), during the perioperative period or during surgery in a
method for maintaining a steady-state hemoglobin concentration in a
patient (WO 95/03068), and as part of a perioperative hemodilution
procedure used prior to surgery in an autologous blood use method
(U.S. Pat. Nos. 5,344,393 and 5,451,205). When a patient suffers a
trauma (i.e., a wound or injury) resulting, for example, from
surgery, an invasive medical procedure, or an accident, the trauma
disturbs the patient's homeostasis. The patient's body biologically
reacts to the trauma to restore homeostasis. This reaction is
referred to herein as a naturally occurring stress response. If the
body's stress response is inadequate or if it occurs well after the
trauma is suffered, the patient is more prone to develop disorders.
III. Reduction of the Oxygen-Affinity of Hemoglobin
The major function of erythrocytes consists in the transport of
molecular oxygen from the lungs to the peripheral tissues. The
erythrocytes contain a high concentration of hemoglobin (30 pg per
cell=35.5 g/100 ml cells) which forms a reversible adduct with
O.sub.2. The O.sub.2 -partial pressure in the lung is about. 100 mm
Hg, in the capillary system is about. 70 mm Hg, against which
O.sub.2 must be dissociated from the oxygenated hemoglobin. Under
physiological conditions, only about 25% of the oxygenated
hemoglobin may be deoxygenated; about. 75% is carried back to the
lungs with the venous blood. Thus, the major fraction of the
hemoglobin-O.sub.2 adduct is not used for the O.sub.2
transport.
Interactions of hemoglobin with allosteric effectors enable an
adaptation to the physiological requirement of maximum O.sub.2
release from the hemoglobin-O.sub.2 adduct with simultaneous
conservation of the highest possible O.sub.2 partial pressure in
the capillary system. 2,3-Diphosphoglycerate increases the
half-saturation pressure of stripped hemoglobin at pH 7.4 from
P(O.sub.2) (1/2)=9.3 mm Hg (37 C.), and 4.3 mm Hg (25 C.) to
P(O.sub.2) (1/2)=23.7 mm Hg (37 C.), and 12.0 mm Hg (25 C.),
respectively (Imai, K. and Yonetani, T. (1975), J. Biol. Chem. 250,
1093-1098). A significantly stronger decrease of the O.sub.2
affinity, i.e., enhancement of the O.sub.2 half-saturation pressure
has been achieved for stripped hemoglobin by binding of inositol
hexaphosphate (phytic acid; IHP) (Ruckpaul, K. et al. (1971)
Biochim. Biophys. Acta 236, 211-221) isolated from vegetal tissues.
Binding of IHP to hemoglobin increases the O.sub.2 half-saturation
pressure to P(O.sub.2) (1/2)=96.4 mm Hg (37 C.), and P(O.sub.2)
(1/2)=48.4 mm Hg (25 C.), respectively. IHP, like
2,3-diphosphoglycerate and other polyphosphates cannot penetrate
the erythrocyte membrane.
Furthermore, the depletion of DPG and ATP in stored red cells leads
to a progressive increase of the oxygen affinity of hemoglobin
contained therein (Balcerzak, S. et al. (1972) Adv. Exp. Med. Biol.
28, 453-447). The O.sub.2 -binding isotherms are measured in the
absence of CO.sub.2 and at constant pH (pH 7.4) in order to
preclude influences of these allosteric effectors on the
half-saturation pressure. The end point of the progressive
polyphosphate depletion is defined by P(O.sub.2) (1/2)=4.2 mm Hg,
which is the half-saturation pressure of totally phosphate-free
(stripped) hemoglobin; the starting point, i.e., P(O.sub.2) (1/2)
of fresh erythrocytes, depends on the composition of the suspending
medium. From these polyphosphate depletion curves a new functional
parameter of stored erythrocytes can be determined, the so-called
half-life time of intra-erythrocytic polyphosphate: 9 d (days) in
isotonic 0.1 M bis-Tris buffer pH 7.4: and 12 d (days) in
acid-citrate-dextrose conservation (ACD) solution.
Several years ago, it was discovered that the antilipidemic drug
clofibric acid lowered the oxygen affinity of hemoglobin solutions
(Abraham et al., J. Med. Chem. 25, 1015 (1982), and Abraham et al.,
Proc. Natl. Acad. Sci USA 80, 324 (1983)). Bezafibrate, another
antilipidemic drug, was later found to be much more effective in
lowering the oxygen affinity of hemoglobin solutions and
suspensions of fresh, intact red cells (Perutz et al., Lancet, 881,
Oct. 15, 1983). Subsequently, X-ray crystallographic studies have
demonstrated that clofibric acid and bezafibrate bind to the same
sites in the central water cavity of deoxyhemoglobin, and that one
bezafibrate molecule will span the sites occupied by two clofibric
acid molecules. Bezafibrate and clofibric acid act by stabilizing
the deoxy structure of hemoglobin, shifting the allosteric
equilibrium toward the low affinity deoxy form. Bezafibrate and
clofibric acid do not bind in any specific manner to either oxy- or
carbonmonoxyhemoglobin.
In more recent investigations, a series of urea derivatives
[2-[4-[[(arylamino)carbonyl]amino]phenoxy]-2-methylpropionic acids]
was discovered that has greater allosteric potency than bezafibrate
at stabilizing the deoxy structure of hemoglobin and shifting the
allosteric equilibrium toward the low oxygen affinity form
(Lalezari, Proc. Natl. Acad. Sci. USA 85, 6117 (1988)).
Drugs which can allosterically modify hemoglobin toward a lower
oxygen affinity state hold potential for many clinical
applications, such as for the treatment of ischemia, shock, and
polycythemia, and as radiosensitizing agents. Unfortunately, the
effects of bezafibrate and the urea derivatives discussed above
have been found to be significantly inhibited by serum albumin, the
major protein in blood serum (Lalezari et al., Biochemistry, 29,
1515 (1990)). Therefore, the clinical usefulness of these drugs is
seriously undermined because in whole blood and in the body, the
drugs would be bound by serum albumin instead of reaching the red
cells, crossing the red cell membrane, and interacting with
hemoglobin protein molecule to produce the desired effect.
There has been considerable interest in medicine, the military
health services, and the pharmaceutical industry in finding methods
to increase blood storage life; to discover radio sensitization
agents; and to develop new blood substitutes. In all these
instances, the availability of either autologous blood or
recombinant Hb solutions is of major interest, provided the oxygen
affinity can be decreased to enhance oxygen delivery to the
tissues.
2,3-Diphosphoglycerate (2,3-DPG) is the normal physiological ligand
for the allosteric site on hemoglobin. However, phosphorylated
inositols are found in the erythrocytes of birds and reptiles.
Specifically, inositol hexaphosphate (IHP), as known as phytic
acid, displaces hemoglobin-bound 2,3-DPG, binding to the allosteric
site with one-thousand times greater affinity. Unfortunately, IHP
is unable to pass unassisted across the erythrocyte membrane.
As emphasized in phase transfer catalysis, it has long been
recognized that organic and inorganic anions can be efficiently
solubilized in organic media when associated to tri- or tetra-alkyl
ammoniums counter-cations. IHP is a highly charged polyphosphate
insoluble in many organic solvents, but is also capable of multiple
ionic bonds with organic ammoniums. Since pKa values of all acidic
protons of IHP have been measured either by NMR (L. R. Isbrandt, R.
P. Oertel J. Am. Chem. Soc. 1980, 102, 3144-48), or by
potentiometric methods (H. Bieth, B. Spiess J. Chem. Soc., Faraday
Transaction 1 1986, 25, 6701-6705), it has been established that
IHP bears 7 or 8 charges at physiological pH. It also means that
IHP can be associated to at least seven lipophilic cations in
physiological conditions. Thus, as a function of the associated
ammoniums, the lipophilicity can theoretically be shifted to a
cell-membrane compatible IHP-complex, the ideal case being a
poly-ammonium IHP derivative that would be soluble in both water
and low polarity media. Because of the emergence of the gene
transfection research field a huge number of cationic lipids are
described in the literature, and extensively reviewed. In most of
the cases these chemical vectors are lipids functionnalized with
amines, poly-amines, (poly-) guanidiniums, and, in rare cases,
phosphoniums. These cationic lipids are designed for gene delivery,
and the mechanism by which they transport oligonucleotides across
biological membranes may be very different for the delivery of a
small poly-anionic molecule such as phytic acid.
Moreover, to avoid the technical problems associated to drug
delivery mediated by liposomes or vesicles, we decided, as a first
approach, to prepare water-soluble lipophilyzed IHP derivatives.
Thus, a first library of IHP-ammoniums salts has been prepared from
commercially available non-lipidic amines in order to assess the
structural parameters allowing both the transport (by increasing
the lipophilicity) and the water solubilities of the salts (for the
improvement of the bioavailability). Both the biological and
physical properties of each salts have been evaluated by the
measurement of O.sub.2 dissociation curves, performed on whole
blood, and 1-octanol/water partition coefficients, respectively. IV
Enhanced Oxygen Delivery in Mammals
The therapy of oxygen deficiencies requires the knowledge of
parameters which characterize both the O.sub.2 transport capacity
and the O.sub.2 release capacity of human RBCs. The parameters of
the O.sub.2 transport capacity, i.e., Hb concentration, the number
of RBCs, and hemocrit, are commonly used in clinical diagnosis.
However, the equally important parameters of the O.sub.2 release
capacity, i.e., O.sub.2 half-saturation pressure of Hb and RBCs,
and the amounts of high and low oxygen affinity hemoglobins in
RBCs, are not routinely determined and were not given serious
consideration until pioneering work by Gerosonde and Nicolau (Blut,
1979, 39, 1-7).
In the 1980s, Nicolau et al. (J. Appl. Physiol. 58:1810-1817
(1985); "PHYTIC ACID: Chemsitry and Applications"; Graf, E., Ed.;
Pilatus Press, Minneapolis, Minn., USA; 1986; and Proc. Natl. Acad.
Sci. USA 1987, 84, 6894-6898) reported that the encapsulation in
red blood cells (RBCs) of IHP, via a technique of controlled lysis
and resealing, results in a significant decrease in the hemoglobin
affinity for oxygen. The procedure yielded RBCs with unchanged life
spans, normal ATP and K+ levels, and normal Theological competence.
Enhancement of the O.sub.2 -release capacity of these cells brought
about significant physiological effects in piglets: 1) reduced
cardiac output, linearly dependent on the P50 value of the RBCs; 2)
increased arteriovenous difference; and 3) improved tissue
oxygenation. Long term experiments showed that in piglets the high
P.sub.50 value of IHP-RBCs was maintained over the entire life
spans of the RBCs.
More recently, Nicolau et al. (TRANSFUSION 1995, 35, 478-486; and
U.S. Pat. No. 5,612,207) reported the use of a large-volume,
continuous-flow electroporation system for the encapsulating IHP in
human RBCs. These modified RBCs possess P.sub.50 values of
approximately 50 torr, roughly twice that of unmodified human RBCs.
Additionally, 85% of the RBCs survived the electroporation process,
displaying hematologic indices nearly identical to those of
unmodified RBCs. Nicolau's electroporation system processes one
unit of blood every ninety minutes. V. Specific Clinical
Applications of Enhanced Oxygen Delivery
There are numerous clinical conditions that would benefit from
treatments that would increase tissue delivery of oxygen bound to
hemoglobin. For example, the leading cause of death in the United
States today is cardiovascular disease. The acute symptoms and
pathology of many cardiovascular diseases, including congestive
heart failure, myocardial infarction, stroke, intermittent
claudication, and sickle cell anemia, result from an insufficient
supply of oxygen in fluids that bathe the tissues. Likewise, the
acute loss of blood following hemorrhage, traumatic injury, or
surgery results in decreased oxygen supply to vital organs. Without
oxygen, tissues at sites distal to the heart, and even the heart
itself, cannot produce enough energy to sustain their normal
functions. The result of oxygen deprivation is tissue death and
organ failure.
Although the attention of the American public has long been focused
on the preventive measures required to alleviate heart disease,
such as exercise, appropriate dietary habits, and moderation in
alcohol consumption, deaths continue to occur at an alarming rate.
Since death results from oxygen deprivation, which in turn results
in tissue destruction and/or organ dysfunction, one approach to
alleviate the life-threatening consequences of cardiovascular
disease is to increase oxygenation of tissues during acute stress.
The same approach is also appropriate for persons suffering from
blood loss or chronic hypoxic disorders, such as congestive heart
failure.
Another condition which could benefit from an increase in the
delivery of oxygen to the tissues is anemia. A significant portion
of hospital patients experience anemia or a low "crit" caused by an
insufficient quantity of red blood cells or hemoglobin in their
blood. This leads to inadequate oxygenation of their tissues and
subsequent complications. Typically, a physician can temporarily
correct this condition by transfusing the patient with units of
packed red blood cells.
Enhanced blood oxygenation may also reduce the number of
heterologous transfusions and allow use of autologous transfusions
in more case. The current method for treatment of anemia or
replacement of blood loss is transfusion of whole human blood. It
is estimated that three to four million patients receive
transfusions in the U.S. each year for surgical or medical needs.
In situations where there is more time it is advantageous to
completely avoid the use of donor or heterologous blood and instead
use autologous blood.
Often the amount of blood which can be drawn and stored prior to
surgery limits the use of autologous blood. Typically, a surgical
patient does not have enough time to donate a sufficient quantity
of blood prior to surgery. A surgeon would like to have several
units of blood available. As each unit requires a period of several
weeks between donations and can not be done less than two weeks
prior to surgery, it is often impossible to sequester an adequate
supply of blood. By processing autologous blood with IHP, less
blood is required and it becomes possible to completely avoid the
transfusion of heterologous blood.
Because IHP-treated RBCs may release up to 2-3 times as much oxygen
as untreated red cells, in many cases, a physician will need to
transfuse fewer units of IHP-treaded red cells. This exposes the
patient to less heterologous blood, decreases the extent of
exposure to vital diseases from blood donors and minimizes immune
function disturbances secondary to transfusions. The ability to
infuse more efficient red blood cells is also advantageous when the
patients blood volume is excessive. In more severe cases, where
oxygen transport is failing, the ability to improve rapidly a
patient's tissue oxygenation is life saving.
Although it is evident that methods of enhancing oxygen delivery to
tissues have potential medical applications, currently there are no
methods clinically available for increasing tissue delivery of
oxygen bound to hemoglobin. Transient, 6 to 12 hour elevations of
oxygen deposition have been described in experimental animals using
either DPG or molecules that are precursors of DPG. The natural
regulation of DPG synthesis in vivo and its relatively short
biological half-life, however, limit the DPG concentration and the
duration of increased tissue PO.sub.2, and thus limit its
therapeutic usefulness.
Additionally, as reported in Genetic Engineering News, Vol. 12, No.
6, Apr. 15, 1992, several groups are attempting to engineer free
oxygen-carrying hemoglobin as a replacement for human blood.
Recombinant, genetically modified human hemoglobin that does not
break down in the body and that can readily release up to 30% of
its bound oxygen is currently being tested by Somatogen, Inc., of
Boulder Colo. While this product could be useful as a replacement
for blood lost in traumatic injury or surgery, it would not be
effective to increase PO.sub.2 levels in ischemic tissue, since its
oxygen release capacity is equivalent to that of natural hemoglobin
(27-30%). As are all recombinant products, this synthetic
hemoglobin is also likely to be a costly therapeutic.
Synthetic human hemoglobin has also been produced in neonatal pigs
by injection of human genes that control hemoglobin production.
This product may be less expensive product than the Somatogen
synthetic hemoglobin, but it does not solve problems with oxygen
affinity and breakdown of hemoglobin in the body.
SUMMARY OF THE INVENTION
The present invention relates to compositions, and methods of use
thereof, consisting essentially of aliphatic ammonium cations and
inositol hexaphosphate (IHP), an allosteric effector of
hemoglobin.
The aliphatic ammonium cation is substituted with one or more times
with aliphatic groups, which can be the same or different. In
certain embodiments, the aliphatic ammonium cation is a primary
ammonium cation represented by the general formula NH.sub.3 (R),
wherein R is an aliphatic group, preferably an alkyl, more
preferably a lower alkyl, i.e., a C.sub.1 -C.sub.6 alkyl, and even
more preferably a C.sub.3 -C.sub.6 cycloalkyl. In certain preferred
embodiments, the ammonium cation is preferably derived from cyclic
amines.
In certain embodiments, the present invention relates to compounds,
and compositions thereof, that deliver IHP into erythrocytes ex
vivo, for lowering the oxygen affinity of hemoglobin in red blood
cell suspensions and whole blood. It is an object of this invention
to provide methods for delivering IHP into erythrocytes in whole
blood, utilizing compounds, or compositions thereof, that do not
lose their effectiveness in the presence of normal concentrations
of the remaining components of whole blood.
In certain embodiments, the present invention relates to a method
of treating a subject for any one or more diseases where an
increase in oxygen delivery of hemoglobin would be of benefit
comprising the steps of treating red blood cells or whole blood ex
vivo with one or more compounds or compositions of the present
invention, followed by suitably purifying said red blood cells or
whole blood, and administering the thus prepared red blood cells or
whole blood to said subject. By `suitably purifying` it is meant a
method of washing and separating, for example by centrifugation,
the red blood cell- or whole blood-allosteric effector suspension
and discarding the supernatant until no non-encapsulated allosteric
effector can be detected. An exemplary method is presented in
detail by Nicolau et al. in U.S. Pat. No. 5,612,207, which is
incorporated by reference herein.
Ligands for the allosteric site of hemoglobin interact with the
hemoglobin molecule and impact its ability to bind oxygen. This
invention is particularly concerned with the delivery of IHP,
causing oxygen to be bound relatively less tightly to hemoglobin,
such that oxygen is off-loaded from the hemoglobin molecule more
easily.
The process of allosterically modifying hemoglobin towards a lower
oxygen affinity state in whole blood may be used in a wide variety
of applications, including treatments for ischemia, heart disease,
wound healing, radiation therapy of cancer, and adult respiratory
distress syndrome (ARDS). Furthermore, a decrease in the oxygen
affinity of hemoglobin in whole blood will extend its useful
shelf-life vis-a-vis transfusions, and/or restore the oxygen
carrying capacity of aged blood.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 tabulates the names or structures of various ammonium salts
of inositol hexaphosphate and the corresponding abbreviations used
herein.
FIG. 2 tabulates the names or structures of various ammonium salts
of inositol hexaphosphate and the corresponding abbreviations used
herein.
FIG. 3 tabulates the names or structures of various ammonium salts
of inositol hexaphosphate and the corresponding abbreviations used
herein.
FIG. 4 tabulates the names, references, molecular formulas, and
molecular weights of various cyclic primary ammonium salts of
inositol hexaphosphate.
FIG. 5 tabulates the names, references, molecular formulas, and
molecular weights of various amino acid and acyclic primary
ammonium salts of inositol hexaphosphate.
FIG. 6 tabulates the names, references, molecular formulas, and
molecular weights of various secondary ammonium salts of inositol
hexaphosphate.
FIG. 7 tabulates the names, references, molecular formulas, and
molecular weights of various tertiary ammonium salts of inositol
hexaphosphate.
FIG. 8 tabulates the names, references, molecular formulas, and
molecular weights of various diammonium salts of inositol
hexaphosphate.
FIG. 9 tabulates the names, references, molecular formulas, and
molecular weights of various tri- and tetra-ammonium salts of
inositol hexaphosphate.
FIG. 10 tabulates the name, reference, molecular formula, and
molecular weight of a hexa-ammonium salt of inositol
hexaphosphate.
FIG. 11 depicts several ammonium salts of IHP and their
corresponding K.sub.ow and K.sub.os partition coefficients.
FIG. 12 compares K.sub.ow dependence on IHP starting concentration
of the IHP salts, SV75 (9 cycloheptylammonium) and SV131 (11
cyclooctylammonium).
FIG. 13 depicts K.sub.os variation as a function of
cyclooctylammonium concentration.
FIG. 14 depicts different IHP derivatives classified by ammonium
types, the number of cations per IHP molecule as well as the
reference number. The P.sub.50 shift values are compared to the
control experiment (underlined), corresponding to a test performed
under the same conditions but with IHP in sodium form.
FIG. 15 tabulates the P.sub.50 values at various osmolarities of
whole blood, and free hemoglobin that has been pre-incubated with
various ammonium salts of inositol hexaphosphate.
FIG. 16 tabulates the P.sub.50 values at various osmolarities of
whole blood, and free hemoglobin that has been pre-incubated with
various ammonium salts of inositol hexaphosphate.
FIG. 17 tabulates the P.sub.50 values at various osmolarities of
whole blood, and free hemoglobin that has been pre-incubated with
various ammonium salts of inositol hexaphosphate.
FIG. 18 depicts how CYCLAM, in a cis- or trans-decaline
conformation may tightly bind 1,2-syn- or
1,2-trans-diphosphates.
FIG. 19 depicts several di-, tri-, and tetra-ammonium salts of IHP
and their corresponding K.sub.ow and K.sub.os partition
coefficients.
FIG. 20 depicts the structures of DPG salts of various ammonium
ions and their 1-octanol/water and 1-octanol/serum partition
coefficients.
DETAILED DESCRIPTION OF THE INVENTION I. Overview
The process of allosterically modifying hemoglobin towards a low
oxygen affinity state in whole blood could be used in a wide
variety of applications including in treatments for ischemia, heart
disease, complications associated with angioplasty, wound healing,
radiation therapy of cancer, adult respiratory distress syndrome
(ARDS), etc., in extending the shelf-life of blood or restoring the
oxygen carrying capacity of out-dated blood, and as sensitizers for
x-ray irradiation in cancer therapy, as well as in many other
applications.
This invention is related to the use of allosteric hemoglobin
modifier compounds in red blood cell suspensions, e.g., in whole
blood. Serum albumin, which is the most abundant protein in blood
plasma, has been identified as inhibiting the allosteric effects of
clofibric acid, bezafibrate, and L3,5/L3,4,5. The precise nature of
this inhibition is not fully understood, but appears to be related
to these compounds binding to the serum albumin. In contrast, the
subject compounds have been found to be relatively unaffected by
the presence of serum albumin. Ligands for the allosteric site of
hemoglobin that are not adversely effected by serum albumin
represent particularly good candidates for drug applications, since
the performance of the drug will not be frustrated by the presence
of serum albumin present in a patient's blood.
This invention relates to the incorporation of a wide variety of
therapeutically useful substances into mammalian red blood cells
(RBCs), which could not previously be accomplished without
unacceptable losses of RBC contents and/or integrity. In
particular, the compounds and methods of the present invention make
possible the introduction or incorporation into RBCs of anionic
agents, such as DNA, RNA, chemotherapeutic agents, and antibiotic
agents. These and other water-soluble substances may be used for a
desired slow continuous delivery or targeted delivery when the
treated and purified RBC carrier is later injected in vivo. The
particular anion or polyanion to be selected can be based on
whether an allosteric effector of hemoglobin would be desirable for
a particular treatment.
The present invention provides a novel method for increasing the
oxygen-carrying capacity of erythrocytes. In accordance with the
method of the present invention, the IHP combines with hemoglobin
in a stable way, and shifts its oxygen releasing capacity.
Erythrocytes with IHP-hemoglobin can release more oxygen per
molecule than hemoglobin alone, and thus more oxygen is available
to diffuse into tissues for each unit of blood that circulates. IHP
is preferably added to red blood cells in vitro or ex vivo, as it
appears that it is toxic to animals under certain
circumstances.
Another advantage of IHP-treated red blood cells is that they show
the Bohr effect in circulation and when stored. Normal red blood
cells that have been stored do not regain their maximum oxygen
carrying capacity in circulation for approximately 24 hours. This
is because the DPG present in normal red blood cells is degraded by
native enzymes, e.g., phosphatases, during storage and must be
replaced by the body after transfusion. In contrast, red blood
cells treated according to the present invention retain their
maximum oxygen carrying capacity during storage and therefore can
deliver oxygen to the tissues in response to demand immediately
after transfusion into a human or animal because there are no
native enzymes in erythrocytes which degrade IHP.
IHP-treated RBCs may be used in the treatment of acute and chronic
conditions, including, but not limited to, hospitalized patients,
cardiovascular operations, chronic anemia, anemia following major
surgery, coronary infarction and associated problems, chronic
pulmonary disease, cardiovascular patients, autologous
transfusions, as an enhancement to packed red blood cells
transfusion (hemorrhage, traumatic injury, or surgery) congestive
heart failure, myocardial infarction (heart attack), stroke,
peripheral vascular disease, intermittent claudication, circulatory
shock, hemorrhagic shock, anemia and chronic hypoxia, respiratory
alkalemia, metabolic alkalosis, sickle cell anemia, reduced lung
capacity caused by pneumonia, surgery, complications associated
with angioplasty, pneumonia, trauma, chest puncture, gangrene,
anaerobic infections, blood vessel diseases such as diabetes,
substitute or complement to treatment with hyperbaric pressure
chambers, intra-operative red cell salvage, cardiac inadequacy,
anoxia-secondary to chronic indication, organ transplant, carbon
monoxide, nitric oxide, and cyanide poisoning.
This invention is related to a method of treating a subject for any
one or more of the above diseases comprising the steps of treating
red blood cells or whole blood ex vivo with one or more compounds
or compositions of the present invention, followed by suitably
purifying said red blood cells or whole blood, and administering
the thus prepared red blood cells or whole blood to said subject.
By `suitably purifying` it is meant a method of washing and
separating the red blood cell- or whole blood-allosteric effector
suspension and discarding the supernatant until no non-encapsulated
allosteric effector can be detected, e.g., as devised by Nicolau et
al. in U.S. Pat. No. 5,612,207. Alternatively, a compound comprised
of an allosteric effector can be administered directly to a subject
if the compound does not have toxic effects in the subject, or at
least its beneficial effects predominate over its toxicity in a
subject. Toxicity of a compound in a subject can be determined
according to methods known in the art.
Treating a human or animal for any one or more of the above disease
states is done by transfusing into the human or animal between
approximately 0.1 and 6 units (1 unit=500 mL) of IHP-treated blood
that has been prepared according to the present invention. In
certain cases, blood exchange with IHP-treated blood may be
possible. The volume of IHP-treated red blood cells that is
administered to the human or animal will depend upon the value of
P.sub.50 for the IHP-treated RBCs. It is to be understood that the
volume of IHP-treated red blood cells that is administered to the
patient can vary and still be effective. IHP-treated RBCs are
similar to normal red blood cells in every respect except that
their P.sub.50 value is shifted towards higher partial pressures of
O.sub.2. Erythrocytes release oxygen only in response to demand by
organs and tissue. Therefore, the compounds, compositions thereof,
and methods of the present invention will only restore a normal
level of oxygenation to healthy tissue, avoiding the cellular
damage that is associated with an over-abundance of oxygen.
Because the compounds, compositions, and methods of the present
invention are capable of allosterically modifying hemoglobin to
favor the low oxygen affinity "T" state (i.e., right shifting the
equilibrium curve), RBC's or whole blood treated with the compounds
of the present invention and subsequently purified will be useful
in treating a variety of disease states in mammals, including
humans, wherein tissues suffer from low oxygen tension, such as
cancer and ischemia. Furthermore, as disclosed by Hirst et al.
(Radiat. Res., 112, (1987), pp. 164), decreasing the oxygen
affinity of hemoglobin in circulating blood has been shown to be
beneficial in the radiotherapy of tumors. RBC's or whole blood
treated with the compounds of the present invention and
subsequently purified may be administered to patients in whom the
affinity of hemoglobin for oxygen is abnormally high. For example,
certain hemoglobinopathies, certain respiratory distress syndromes,
e.g., respiratory distress syndromes in new born infants aggravated
by high fetal hemoglobin levels, and conditions in which the
availability of hemoglobin/ oxygen to the tissues is decreased
(e.g., in ischemic conditions such as peripheral vascular disease,
coronary occlusion, cerebral vascular accidents, or tissue
transplant). The compounds and compositions may also be used to
inhibit platelet aggregation, antithrombotic purposes, and wound
healing.
Additionally, the compounds and compositions of the present
invention can be added to whole blood or packed cells preferably at
the time of storage or at the time of transfusion in order to
facilitate the dissociation of oxygen from hemoglobin and improve
the oxygen delivering capability of the blood. When blood is
stored, the hemoglobin in the blood tends to increase its affinity
for oxygen by losing 2,3-diphosphoglycerides. As described above,
the compounds and compositions of this invention are capable of
reversing and/or preventing the functional abnormality of
hemoglobin observed when whole blood or packed cells are stored.
The compounds and compositions may be added to whole blood or red
blood cell fractions in a closed system using an appropriate
reservoir in which the compound or composition is placed prior to
storage or which is present in the anticoagulating solution in the
blood collecting bag.
Administration to a patient can be achieved by intravenous or
intraperitoneal injection where the dose of treated red blood cells
or whole blood and the dosing regiment is varied according to
individual's sensitivity and the type of disease state being
treated.
Solid tumors are oxygen deficient masses. The compounds,
compositions and methods of this invention may be exploited to
cause more oxygen to be delivered to tumors, increasing radical
formation and thereby increasing tumor killing during radiation. In
this context, such IHP-treated blood will only be used in
conjunction with radiotherapy.
The compounds, compositions and methods of this invention may be
exploited to cause more oxygen to be delivered at low blood flow
and low temperatures, providing the ability to decrease or prevent
the cellular damage, e.g., myocardial or neuronal, typically
associated with these conditions.
The compounds, compositions and methods of this invention may be
exploited to decrease the number of red blood cells required for
treating hemorrhagic shock by increasing the efficiency with which
they deliver oxygen.
Damaged tissues heal faster when there is better blood flow and
increased oxygen tension. Therefore, the compounds, compositions
and methods of this invention may be exploited to speed wound
healing. Furthermore, by increasing oxygen delivery to wounded
tissue, the compounds, compositions and methods of this invention
may play a role in the destruction of infection causing bacteria at
a wound.
The compounds, compositions and methods of this invention may be
effective in enhancing the delivery oxygen to the brain, especially
before complete occlusion and reperfusion injuries occur due to
free radical formation. Furthermore, the compounds, compositions
and methods of this invention of this invention should reduce the
expansion of arterioles under both hypoxic and hypotensive
conditions.
The compounds, compositions and methods of this invention of this
invention should be capable of increasing oxygen delivery to
blocked arteries and surrounding muscles and tissues, thereby
relieving the distress of angina attacks.
Acute respiratory disease syndrome (ARDS) is characterized by
interstitial and/or alveolar edema and hemorrhage as well as
perivascular lung edema associated with the hyaline membrane,
proliferation of collagen fibers, and swollen epithelium with
increased pinocytosis. The enhanced oxygen delivering capacity
provided to RBCs by the compounds, compositions and methods of this
invention could be used in the treatment and prevention of ARDS by
militating against lower than normal oxygen delivery to the
lungs.
There are several aspects of cardiac bypass surgery that make
attractive the use of compounds or compositions or methods of the
present invention. First, the compounds and compositions of the
present invention may act as neuroprotective agents. After cardiac
bypass surgery, up to 50-70% of patients show some signs of
cerebral ischemia based on tests of cognitive function. Up to 5% of
these patients have evidence of stroke. Second, cardioplegia is the
process of stopping the heart and protecting the heart from
ischemia during heart surgery. Cardioplegia is performed by
perfusing the coronary vessels with solutions of potassium chloride
and bathing the heart in ice water. However, blood cardioplegia is
also used. This is where potassium chloride is dissolved in blood
instead of salt water. During surgery the heart is deprived of
oxygen and the cold temperature helps slow down metabolism.
Periodically during this process, the heart is perfused with the
cardioplegia solution to wash out metabolites and reactive species.
Cooling the blood increases the oxygen affinity of its hemoglobin,
thus making oxygen unloading less efficient. However, treatment of
blood cardioplegia with RBC's or whole blood previously treated
with compounds or compositions of the present invention and
subsequently purified will counteract the effects of cold on oxygen
affinity and make oxygen release to the ischemic myocardium more
efficient, possibly improving cardiac function after the heart
begins to beat again. Third, during bypass surgery the patient's
blood is diluted for the process of pump prime. This hemodilution
is essentially acute anemia. Because the compounds and compositions
of the present invention make oxygen transport more efficient,
their use during hemodilution (whether in bypass surgery or other
surgeries, such as orthopedic or vascular) would enhance
oxygenation of the tissues in an otherwise compromised condition.
Additionally, the compounds and methods of the present invention
will also find use in patients undergoing angioplasty, who may
experience acute ischemic insult, e.g., due to the dye(s) used in
this procedure.
Additionally, microvascular insufficiency has been proposed by a
number of investigators as a possible cause of diabetic neuropathy.
The interest in microvascular derangement in diabetic neuropathic
patients has arisen from studies suggesting that absolute or
relative ischemia may exist in the nerves of diabetic subjects due
to altered function of the endo- and/or epineurial blood vessels.
Histopathologic studies have shown the presence of different
degrees of endoneurial and epineurial microvasculopathy, mainly
thickening of blood vessel wall or occlusion. A number of
functional disturbances have also been demonstrated in the
microvasculature of the nerves of diabetic subjects. Studies have
demonstrated decreased neural blood flow, increased vascular
resistance, decreased pO.sub.2 and altered vascular permeability
characteristics such as a loss of the anionic charge barrier and
decreased charge selectivity. Abnormalities of cutaneous blood flow
correlate with neuropathy, suggesting that there is a clinical
counterpart to the microvascular insufficiency that may prove to be
a simple non-invasive test of nerve fiber dysfunction. Accordingly,
patients suffering from diabetic neuropathies and/or other
neurodegenerative disorders will likely benefit from treatment
based on the compounds and methods of the present invention.
Red blood cells or whole blood previously treated with the
compounds of the present invention and subsequently suitably
purified may be used to enhance oxygen delivery in any organism,
e.g., fish, that uses a hemoglobin with an allosteric binding site.
II. Definitions
For convenience, certain terms employed in the specification,
examples, and appended claims are collected here. As used
throughout this specification and the claims, the following terms
have the following meanings:
The term "hemoglobin" includes all naturally- and
non-naturally-occurring hemoglobin.
The term "hemoglobin preparation" includes hemoglobin in a
physiologically compatible carrier or lyophilized hemoglobin
reconstituted with a physiologically compatible carrier, but does
not include whole blood, red blood cells or packed red blood
cells.
The term "toxic" refers to a property where the deleterious effects
are greater than the beneficial effects.
The term "nontoxic" refers to a property where the beneficial
effects are greater than the deleterious effects.
The term "whole blood" refers to blood containing all its natural
constituents, components, or elements or a substantial amount of
the natural constituents, components, or elements. For example, it
is envisioned that some components may be removed by the
purification process before administering the blood to a
subject.
"Purified", "purification process", and "purify" all refer to a
state or process of removing one or more compounds of the present
invention from the red blood cells or whole blood such that when
administered to a subject the red blood cells or whole blood is
nontoxic.
"Non-naturally-occurring hemoglobin" includes synthetic hemoglobin
having an amino-acid sequence different from the amino-acid
sequence of hemoglobin naturally existing within a cell, and
chemically-modified hemoglobin. Such non-naturally-occurring mutant
hemoglobin is not limited by its method of preparation, but is
typically produced using one or more of several techniques known in
the art, including, for example, recombinant DNA technology,
transgenic DNA technology, protein synthesis, and other
mutation-inducing methods.
"Chemically-modified hemoglobin" is a natural or non-natural
hemoglobin molecule which is bonded to another chemical moiety. For
example, a hemoglobin molecule can be bonded to
pyridoxal-5'-phosphate, or other oxygen-affinity-modifying moiety
to change the oxygen-binding characteristics of the hemoglobin
molecule, to crosslinking agents to form crosslinked or polymerized
hemoglobin, or to conjugating agents to form conjugated
hemoglobin.
"Oxygen affinity" means the strength of binding of oxygen to a
hemoglobin molecule. High oxygen affinity means hemoglobin does not
readily release its bound oxygen molecules. The P50 is a measure of
oxygen affinity.
"Cooperativity" refers to the sigmoidal oxygen-binding curve of
hemoglobin, i.e., the binding of the first oxygen to one subunit
within the tetrameric hemoglobin molecule enhances the binding of
oxygen molecules to other unligated subunits. It is conveniently
measured by the Hill coefficient (n[max]). For Hb A,
n[max]=3.0.
The term "treatment" is intended to encompass also prophylaxis,
therapy and cure.
"Ischemia" means a temporary or prolonged lack or reduction of
oxygen supply to an organ or skeletal tissue. Ischemia can be
induced when an organ is transplanted, or by conditions such as
septic shock and sickle cell anemia.
"Skeletal tissue" means the substance of an organic body of a
skeletal organism consisting of cells and intercellular material,
including but not limited to epithelium, the connective tissues
(including blood, bone and cartilage), muscle tissue, and nerve
tissue.
"Ischemic insult" means damage to an organ or skeletal tissue
caused by ischemia.
"Subject" means any living organism, including humans, and
mammals.
The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticulare, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
As used herein, the term "surgery" refers to the treatment of
diseases, injuries, and deformities by manual or operative methods.
Common surgical procedures include, but are not limited to,
abdominal, aural, bench, cardiac, cineplastic, conservative,
cosmetic, cytoreductive, dental, dentofacial, general, major,
minor, Moh's, open heart, organ transplantation, orthopedic,
plastic, psychiatric, radical, reconstructive, sonic, stereotactic,
structural, thoracic, and veterinary surgery. The method of the
present invention is suitable for patients that are to undergo any
type of surgery dealing with any portion of the body, including but
not limited to those described above, as well as any type of any
general, major, minor, or minimal invasive surgery.
"Minimally invasive surgery" involves puncture or incision of the
skin, or insertion of an instrument or foreign material into the
body. Non-limiting examples of minimal invasive surgery include
arterial or venous catheterization, transurethral resection,
endoscopy (e.g., laparoscopy, bronchoscopy, uroscopy,
pharyngoscopy, cystoscopy, hysteroscopy, gastroscopy, coloscopy,
colposcopy, celioscopy, sigmoidoscopy, and orthoscopy), and
angioplasty (e.g., balloon angioplasty, laser angioplasty, and
percutaneous transluminal angioplasty).
The term "ED.sub.50 " means the dose of a drug that produces 50% of
its maximum response or effect. Alternatively, the dose that
produces a pre-determined response in 50% of test subjects or
preparations.
The term "LD.sub.50 " means the dose of a drug that is lethal in
50% of test subjects.
The term "therapeutic index" refers to the therapeutic index of a
drug defined as LD.sub.50 /ED.sub.50.
The phrases "systemic administration," "administered systemically,"
"peripheral administration" and "administered peripherally" as used
herein mean the administration of a compound, drug or other
material other than directly into the central nervous system, such
that it enters the patient's system and, thus, is subject to
metabolism and other like processes, for example, subcutaneous
administration.
The term "structure-activity relationship (SAR)" refers to the way
in which altering the molecular structure of drugs alters their
interaction with a receptor, enzyme, etc.
The term "ammonium cation" refers to the structure below:
##STR1##
wherein R represents independently for each occurrence H or a
substituted or unsubstituted aliphatic group. An "aliphatic
ammonium cation" refers to the above structure when at least one R
is an aliphatic group. A "quaternary ammonium cation" refers to the
above structure when all four occurrences of R independently
represent aliphatic groups. R can be the same for two or more
occurrences, or different for all four.
The term "heteroatom" as used herein means an atom of any element
other than carbon or hydrogen. Preferred heteroatoms are boron,
nitrogen, oxygen, phosphorus, sulfur and selenium.
The term "electron-withdrawing group" is recognized in the art, and
denotes the tendency of a substituent to attract valence electrons
from neighboring atoms, i.e., the substituent is electronegative
with respect to neighboring atoms. A quantification of the level of
electron-withdrawing capability is given by the Hammett sigma
(.sigma.) constant. This well known constant is described in many
references, for instance, J. March, Advanced Organic Chemistry,
McGraw Hill Book Company, New York, (1977 edition) pp. 251-259. The
Hammett constant values are generally negative for electron
donating groups (.sigma.[P]=-0.66 for NH.sub.2) and positive for
electron withdrawing groups (.sigma.[P]=0.78 for a nitro group),
.sigma.[P] indicating para substitution. Exemplary
electron-withdrawing groups include nitro, acyl, formyl, sulfonyl,
trifluoromethyl, cyano, chloride, and the like. Exemplary
electron-donating groups include amino, methoxy, and the like.
The term "alkyl" refers to the radical of saturated aliphatic
groups, including straight-chain alkyl groups, branched-chain alkyl
groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl
groups, and cycloalkyl substituted alkyl groups. In preferred
embodiments, a straight chain or branched chain alkyl has 30 or
fewer carbon atoms in its backbone (e.g., C.sub.1 -C.sub.30 for
straight chain, C.sub.3 -C.sub.30 for branched chain), and more
preferably 20 or fewer. Likewise, preferred cycloalkyls have from
3-10 carbon atoms in their ring structure, and more preferably have
5, 6 or 7 carbons in the ring structure.
Moreover, the term "alkyl" (or "lower alkyl") as used throughout
the specification, examples, and claims is intended to include both
"unsubstituted alkyls" and "substituted alkyls", the latter of
which refers to alkyl moieties having substituents replacing a
hydrogen on one or more carbons of the hydrocarbon backbone. Such
substituents can include, for example, a halogen, a hydroxyl, a
carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an
acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a
thioformate), an alkoxyl, a phosphoryl, a phosphonate, a
phosphinate, an amino, an amido, an amidine, an imine, a cyano, a
nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a
sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl,
an aralkyl, or an aromatic or heteroaromatic moiety. It will be
understood by those skilled in the art that the moieties
substituted on the hydrocarbon chain can themselves be substituted,
if appropriate. For instance, the substituents of a substituted
alkyl may include substituted and unsubstituted forms of amino,
azido, imino, amido, phosphoryl (including phosphonate and
phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl
and sulfonate), and silyl groups, as well as ethers, alkylthios,
carbonyls (including ketones, aldehydes, carboxylates, and esters),
--CF.sub.3, --CN and the like. Exemplary substituted alkyls are
described below. Cycloalkyls can be further substituted with
alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls,
carbonyl-substituted alkyls, --CF.sub.3, --CN, and the like.
The term "aralkyl", as used herein, refers to an alkyl group
substituted with an aryl group (e.g., an aromatic or heteroaromatic
group).
The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic
groups analogous in length and possible substitution to the alkyls
described above, but that contain at least one double or triple
bond respectively.
Unless the number of carbons is otherwise specified, "lower alkyl"
as used herein means an alkyl group, as defined above, but having
from one to ten carbons, more preferably from one to six carbon
atoms in its backbone structure. Likewise, "lower alkenyl" and
"lower alkynyl" have similar chain lengths. Preferred alkyl groups
are lower alkyls. In preferred embodiments, a substituent
designated herein as alkyl is a lower alkyl.
The term "aryl" as used herein includes 5-, 6- and 7-membered
single-ring aromatic groups that may include from zero to four
heteroatoms, for example, benzene, pyrrole, furan, thiophene,
imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,
pyrazine, pyridazine and pyrimidine, and the like. Those aryl
groups having heteroatoms in the ring structure may also be
referred to as "aryl heterocycles" or "heteroaromatics." The
aromatic ring can be substituted at one or more ring positions with
such substituents as described above, for example, halogen, azide,
alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,
amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,
carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,
ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic
moieties, --CF.sub.3, --CN, or the like. The term "aryl" also
includes polycyclic ring systems having two or more cyclic rings in
which two or more carbons are common to two adjoining rings (the
rings are "fused rings") wherein at least one of the rings is
aromatic, e.g., the other cyclic rings can be cycloalkyls,
cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
The terms ortho, meta and para apply to 1,2-, 1,3- and
1,4-disubstituted benzenes, respectively. For example, the names
1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.
The terms "heterocyclyl" or "heterocyclic group" refer to 3- to
10-membered ring structures, more preferably 3- to 7-membered
rings, whose ring structures include one to four heteroatoms.
Heterocycles can also be polycycles. Heterocyclyl groups include,
for example, thiophene, thianthrene, furan, pyran, isobenzofuran,
chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole,
isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine,
indolizine, isoindole, indole, indazole, purine, quinolizine,
isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline,
quinazoline, cinnoline, pteridine, carbazole, carboline,
phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,
phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine,
oxolane, thiolane, oxazole, piperidine, piperazine, morpholine,
lactones, lactams such as azetidinones and pyrrolidinones, sultams,
sultones, and the like. The heterocyclic ring can be substituted at
one or more positions with such substituents as described above, as
for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,
ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, --CF.sub.3, --CN, or the like.
The terms "polycyclyl" or "polycyclic group" refer to two or more
rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls
and/or heterocyclyls) in which two or more carbons are common to
two adjoining rings, e.g., the rings are "fused rings". Rings that
are joined through non-adjacent atoms are termed "bridged" rings.
Each of the rings of the polycycle can be substituted with such
substituents as described above, as for example, halogen, alkyl,
aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,
sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,
carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde,
ester, a heterocyclyl, an aromatic or heteroaromatic moiety,
--CF.sub.3, --CN, or the like.
The term "carbocycle", as used herein, refers to an aromatic or
non-aromatic ring in which each atom of the ring is carbon.
As used herein, the term "nitro" means --NO.sub.2 ; the term
"halogen" designates --F, --Cl, --Br or --I; the term "sulfhydryl"
means --SH; the term "hydroxyl" means --OH; and the term "sulfonyl"
means --SO.sub.2 --.
The terms "amine" and "amino" are art-recognized and refer to both
unsubstituted and substituted amines, e.g., a moiety that can be
represented by the general formula: ##STR2##
wherein R.sub.9, R.sub.10 and R'.sub.10 each independently
represent a hydrogen, an alkyl, an alkenyl, --(CH.sub.2).sub.m
--R.sub.8, or R.sub.9 and R.sub.10 taken together with the N atom
to which they are attached complete a heterocycle having from 4 to
8 atoms in the ring structure; R.sub.8 represents an aryl; a
cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is
zero or an integer in the range of 1 to 8. In preferred
embodiments, only one of R.sub.9 or R.sub.10 can be a carbonyl,
e.g., R.sub.9, R.sub.10 and the nitrogen together do not form an
imide. In even more preferred embodiments, R.sub.9 and R.sub.10
(and optionally R'.sub.10) each independently represent a hydrogen,
an alkyl, an alkenyl, or --(CH.sub.2).sub.m --R.sub.8. Thus, the
term "alkylamine" as used herein means an amine group, as defined
above, having a substituted or unsubstituted alkyl attached
thereto, i.e., at least one of R.sub.9 and R.sub.10 is an alkyl
group.
The term "acylamino" is art-recognized and refers to a moiety that
can be represented by the general formula: ##STR3##
wherein R.sub.9 is as defined above, and R'.sub.11 represents a
hydrogen, an alkyl, an alkenyl or --(CH.sub.2).sub.m --R.sub.8,
where m and R.sub.8 are as defined above.
The term "amido" is art recognized as an amino-substituted carbonyl
and includes a moiety that can be represented by the general
formula: ##STR4##
wherein R.sub.9, R.sub.10 are as defined above. Preferred
embodiments of the amide will not include imides which may be
unstable.
The term "alkylthio" refers to an alkyl group, as defined above,
having a sulfur radical attached thereto. In preferred embodiments,
the "alkylthio" moiety is represented by one of --S-alkyl,
--S-alkenyl, --S-alkynyl, and --S--(CH.sub.2).sub.m --R.sub.8,
wherein m and R.sub.8 are defined above. Representative alkylthio
groups include methylthio, ethyl thio, and the like.
The term "carbonyl" is art recognized and includes such moieties as
can be represented by the general formula: ##STR5##
wherein X is a bond or represents an oxygen or a sulfur, and
R.sub.11 represents a hydrogen, an alkyl, an alkenyl,
--(CH.sub.2).sub.m --R.sub.8 or a pharmaceutically acceptable salt,
R'.sub.11 represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.2).sub.m --R.sub.8, where m and R.sub.8 are as defined
above. Where X is an oxygen and R.sub.11 or R'.sub.11 is not
hydrogen, the formula represents an "ester". Where X is an oxygen,
and R.sub.11 is as defined above, the moiety is referred to herein
as a carboxyl group, and particularly when R.sub.11 is a hydrogen,
the formula represents a "carboxylic acid". Where X is an oxygen,
and R'.sub.11 is hydrogen, the formula represents a "formate". In
general, where the oxygen atom of the above formula is replaced by
sulfur, the formula represents a "thiolcarbonyl" group. Where X is
a sulfur and R.sub.11 or R'.sub.11 is not hydrogen, the formula
represents a "thiolester." Where X is a sulfur and R.sub.11 is
hydrogen, the formula represents a "thiolcarboxylic acid." Where X
is a sulfur and R.sub.11 ' is hydrogen, the formula represents a
"thiolformate." On the other hand, where X is a bond, and R.sub.11
is not hydrogen, the above formula represents a "ketone" group.
Where X is a bond, and R.sub.11 is hydrogen, the above formula
represents an "aldehyde" group.
The terms "alkoxyl" or "alkoxy" as used herein refers to an alkyl
group, as defined above, having an oxygen radical attached thereto.
Representative alkoxyl groups include methoxy, ethoxy, propyloxy,
tert-butoxy and the like. An "ether" is two hydrocarbons covalently
linked by an oxygen. Accordingly, the substituent of an alkyl that
renders that alkyl an ether is or resembles an alkoxyl, such as can
be represented by one of --O-alkyl, --O-alkenyl, --O-alkynyl,
--O--(CH.sub.2).sub.m --R.sub.8, where m and R.sub.8 are described
above.
The term "sulfonate" is art recognized and includes a moiety that
can be represented by the general formula: ##STR6##
in which R.sub.41 is an electron pair, hydrogen, alkyl, cycloalkyl,
or aryl.
The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized
and refer to trifluoromethanesulfonyl, p-toluenesulfonyl,
methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively.
The terms triflate, tosylate, mesylate, and nonaflate are
art-recognized and refer to trifluoromethanesulfonate ester,
p-toluenesulfonate ester, methanesulfonate ester, and
nonafluorobutanesulfonate ester functional groups and molecules
that contain said groups, respectively.
The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,
ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,
p-toluenesulfonyl and methanesulfonyl, respectively. A more
comprehensive list of the abbreviations utilized by organic
chemists of ordinary skill in the art appears in the first issue of
each volume of the Journal of Organic Chemistry; this list is
typically presented in a table entitled Standard List of
Abbreviations. The abbreviations contained in said list, and all
abbreviations utilized by organic chemists of ordinary skill in the
art are hereby incorporated by reference.
The term "sulfate" is art recognized and includes a moiety that can
be represented by the general formula: ##STR7##
in which R.sub.41 is as defined above.
The term "sulfonamido" is art recognized and includes a moiety that
can be represented by the general formula: ##STR8##
in which R.sub.9 and R'.sub.11 are as defined above.
The term "sulfamoyl" is art-recognized and includes a moiety that
can be represented by the general formula: ##STR9##
in which R.sub.9 and R.sub.10 are as defined above.
The term "sulfonyl", as used herein, refers to a moiety that can be
represented by the general formula: ##STR10##
in which R.sub.44 is selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl,
or heteroaryl.
The term "sulfoxido" as used herein, refers to a moiety that can be
represented by the general formula: ##STR11##
in which R.sub.44 is selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,
aralkyl, or aryl.
A "phosphoryl" can in general be represented by the formula:
##STR12##
wherein Q.sub.1 represented S or O, and R.sub.46 represents
hydrogen, a lower alkyl or an aryl. When used to substitute, e.g.,
an alkyl, the phosphoryl group of the phosphorylalkyl can be
represented by the general formula: ##STR13##
wherein Q.sub.1 represented S or O, and each R.sub.46 independently
represents hydrogen, a lower alkyl or an aryl, Q.sub.2 represents
O, S or N. When Q.sub.1 is an S, the phosphoryl moiety is a
"phosphorothioate".
Analogous substitutions can be made to alkenyl and alkynyl groups
to produce, for example, aminoalkenyls, aminoalkynyls,
amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls,
thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or
alkynyls.
As used herein, the definition of each expression, e.g. alkyl, m,
n, etc., when it occurs more than once in any structure, is
intended to be independent of its definition elsewhere in the same
structure.
It will be understood that "substitution" or "substituted with"
includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, etc.
As used herein, the term "substituted" is contemplated to include
all permissible substituents of organic compounds. In a broad
aspect, the permissible substituents include acyclic and cyclic,
branched and unbranched, carbocyclic and heterocyclic, aromatic and
nonaromatic substituents of organic compounds. Illustrative
substituents include, for example, those described herein above.
The permissible substituents can be one or more and the same or
different for appropriate organic compounds. For purposes of this
invention, the heteroatoms such as nitrogen may have hydrogen
substituents and/or any permissible substituents of organic
compounds described herein which satisfy the valences of the
heteroatoms. This invention is not intended to be limited in any
manner by the permissible substituents of organic compounds.
The phrase "protecting group" as used herein means temporary
substituents which protect a potentially reactive functional group
from undesired chemical transformations. Examples of such
protecting groups include esters of carboxylic acids, silyl ethers
of alcohols, and acetals and ketals of aldehydes and ketones,
respectively. The field of protecting group chemistry has been
reviewed (Greene, T. W.; Wuts, P. G. M Protective Groups in Organic
Synthesis, 2.sup.nd ed.; Wiley: New York, 1991).
Certain compounds of the present invention may exist in particular
geometric or stereoisomeric forms. The present invention
contemplates all such compounds, including cis- and trans-isomers,
R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the
racemic mixtures thereof, and other mixtures thereof, as falling
within the scope of the invention. Additional asymmetric carbon
atoms may be present in a substituent such as an alkyl group. All
such isomers, as well as mixtures thereof, are intended to be
included in this invention.
If, for instance, a particular enantiomer of a compound of the
present invention is desired, it may be prepared by asymmetric
synthesis, or by derivation with a chiral auxiliary, where the
resulting diastereomeric mixture is separated and the auxiliary
group cleaved to provide the pure desired enantiomers.
Alternatively, where the molecule contains a basic functional
group, such as amino, or an acidic functional group, such as
carboxyl, diastereomeric salts are formed with an appropriate
optically-active acid or base, followed by resolution of the
diastereomers thus formed by fractional crystallization or
chromatographic means well known in the art, and subsequent
recovery of the pure enantiomers.
Contemplated equivalents of the compounds described above include
compounds which otherwise correspond thereto, and which have the
same general properties thereof, wherein one or more simple
variations of substituents are made which do not adversely affect
the efficacy of the compound. In general, the compounds of the
present invention may be prepared by the methods illustrated in the
general reaction schemes as, for example, described below, or by
modifications thereof, using readily available starting materials,
reagents and conventional synthesis procedures. In these reactions,
it is also possible to make use of variants which are in themselves
known, but are not mentioned here.
For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87,
inside cover. Also for purposes of this invention, the term
"hydrocarbon" is contemplated to include all permissible compounds
having at least one hydrogen and one carbon atom. In a broad
aspect, the permissible hydrocarbons include acyclic and cyclic,
branched and unbranched, carbocyclic and heterocyclic, aromatic and
nonaromatic organic compounds which can be substituted or
unsubstituted. III. Compounds of the Invention
Several years ago, it was discovered that the antilipidemic drug
clofibric acid lowered the oxygen affinity of hemoglobin solutions
(Abraham et al., J. Med. Chem. 25, 1015 (1982), and Abraham et al.,
Proc. Natl. Acad. Sci. USA 80, 324 (1983)). Bezafibrate, another
antilipidemic drug, was later found to be much more effective in
lowering the oxygen affinity of hemoglobin solutions and
suspensions of fresh, intact red cells (Perutz et al., Lancet, 881,
Oct. 15, 1983). Subsequently, X-ray crystallographic studies have
demonstrated that clofibric acid and bezafibrate bind to the same
sites in the central water cavity of deoxyhemoglobin, and that one
bezafibrate molecule will span the sites occupied by two clofibric
acid molecules. Bezafibrate and clofibric acid act by stabilizing
the deoxy structure of hemoglobin, shifting the allosteric
equilibrium toward the low affinity deoxy form. Bezafibrate and
clofibric acid do not bind in any specific manner to either oxy- or
carbonmonoxyhemoglobin.
In later investigations, a series of urea derivatives
[2-[4-[[(arylamino)carbonyl]-amino]phenoxy]-2-methylpropionic
acids] was discovered that has greater allosteric potency than
bezafibrate at stabilizing the deoxy structure of hemoglobin and
shifting the allosteric equilibrium toward the low oxygen affinity
form (Lalezari, Proc. Natl. Acad. Sci. USA 85, 6117 (1988)).
It has been determined that certain allosteric hemoglobin modifier
compounds are hydrophobic molecules that can be bound to the body's
neutral fat deposits and lipophilic receptors sites, thus lowering
their potency due to a decreased concentration in RBCs.
Administration of a hydrophobic compound, such as a mixture of
anesthetic molecules, will saturate the body's neutral fat deposits
and lipophilic receptor sites, and thereby increase the
concentration of this type of allosteric modifiers in RBCs, where
higher concentrations of effector will increase its ability to
interact with hemoglobin, causing delivery of more oxygen.
Ligands for the allosteric site of hemoglobin, also known as
allosteric effectors of hemoglobin, include 2,3-diphosphoglycerate
(DPG), inositol hexakisphosphate (IHP), bezafibrate (Bzf), LR16 and
L35 (two recently synthesized derivatives of Bzf), and pyridoxal
phosphate. Additionally, hemoglobin's affinity for oxygen can be
modulated through electrostatic interactions with chloride and/or
organophosphate anions present in RBCs. These effectors, which bind
preferentially to the deoxy-Hb tetramers at a distance from the
heme groups, play a major role in the adaptation of the respiratory
properties of hemoglobin to either allometric-dependent oxygen
needs or to various hypoxic environments. Additionally, protons and
carbon dioxide are physiological regulators for the oxygen affinity
of hemoglobin. The heterotropic allosteric interaction between the
non-heme ligands and oxygen, collectively called the Bohr effect,
facilitates not only the transport of oxygen but also the exchange
of carbon dioxide.
The present invention relates to compositions, and methods of use
thereof, consisting essentially of a nontoxic ammonia cation
(preferably water-soluble), and inositol hexaphosphate (IHP). In
certain embodiments, the nontoxic ammonium cation is represented by
the general formula N(R).sub.4, wherein R is, independently for
each occurrence, H or an aliphatic group, which aliphatic group is
preferably an alkyl, more preferably a lower (C1-C6) alkyl, and
even more preferably a C1-C10 cyclic alkyl. In certain preferred
embodiments, the ammonium cation is preferably derived from cyclic
organic bases.
In certain embodiments, the present invention is related to
compounds, and compositions thereof, which deliver IHP into
erythrocytes ex vivo. Additionally, the invention is directed to
the use of the compounds or compositions thereof that are effective
in delivering IHP into erythrocytes, lowering the oxygen affinity
state in red blood cell suspensions and whole blood. It is an
object of this invention to provide methods for delivering IHP into
erythrocytes in whole blood, utilizing compounds or compositions
thereof that do not lose their effectiveness in the presence of
normal concentrations of the remaining components of whole
blood.
In certain embodiments, the present invention is related to a
method of treating red blood cells or whole blood ex vivo with one
or more nontoxic compounds or compositions of the present
invention, suitably purifying said red blood cells or whole blood,
and administering said purified red blood cells or whole blood to a
subject for any treatment where an increase in oxygen delivery by
hemoglobin would be a benefit.
In part, the present invention is directed toward the design of
water-soluble membrane compatible molecules comprising ammonium
cationic moieties, e.g., lipophilic ammonium groups. These
molecules form complexes with IHP; such complexes are useful for
the deliver of IHP into the cytoplasm of erythrocytes.
The ammonium group of the cationic component of the compounds of
the present invention is particularly well suited for interaction
with the phosphate residues of IHP and congeners thereof because of
the coulombic interactions, i.e., the attraction between opposite
charges, that can be established between the two moieties. We
report here the use of ammonium salts for the efficient delivery of
IHP into mammalian erythrocytes. Our data demonstrate the
usefulness, convenience, and versatility of ammonium salts for
delivery of IHP into the cytoplasm of mammalian cells.
In certain embodiments, the compounds of the present invention are
represented by generalized structure 1: ##STR14##
wherein nC.sup.+ represents nona-cyclohexylammonium-tri-sodium,
bis-dicyclohexylammonium-deca-sodium, octa-dicyclohexylammonium,
hepta-1-aza-3-hydroxyl-bicyclo[2.2.2]cyclooctanium,
dodeca-1-aza-3-hydroxyl-bicyclo[2.2.2]cyclooctanium,
nona-piperidinium, penta-H.sub.3 N-Phe-OMe, nona-H.sub.3 N-Phe-OMe,
hexa-1-indanylammonium, hepta-2-norbornylammonium,
nona-decahydroquinolinium, hepta-H.sub.3 N-Phe-OEt, hexa-H.sub.3
N-Phe-OEt, octa-H.sub.3 N-sec-Leu-Ot-Bu,
dodeca-diisopropylammonium, octa-H.sub.3 N-Pro-Ot-Bu, deca-H.sub.3
N-Tyr-OEt, tetra-cyclohexyl-1,2-bis-ammonium,
nona-cycloheptylammonium, undecacyclopentylammonium,
undecacyclohexylammonium, penta-(N,N'-dibenzyl)-ethylenediammonium,
octa menthyl-1,8-diammonium, penta
cyclohexyl-(1,3-bismethylammonium), penta
(.+-.)-(1,2-trans-diphenyl)-ethylenediammonium, nona
N-cyclohexyl-piperidinium, bis (N.sup.1,N.sup.3
-cyclohexyl)-dipropylenetriammonium, tris
tri-(N-cyclohexyl-2-amino-ethyl)-ammonium, tetra
N,N'-di-(3-(N-cyclohexyl-amino)-propyl)-piperazinium, tris
tri-(N-cycloheptyl-2-amino-ethyl)-ammonium, tri
N,N'-di-(3-(N-cyclooctyl-amino)-propyl)-piperazinium, or bis
N,N',N",N'"-tetrahexyl-cyclam; and A.sup.n- represents a conjugate
base of inositol hexaphosphate, wherein n equals the number of
cations comprised by nC.sup.+.
In certain embodiments, the present invention relates to a
pharmaceutical composition, comprising a nontoxic compound of the
present invention; and a pharmaceutically acceptable excipient. IV.
IHP-ammonium salts Preparation
IHP ammonium salts can be efficiently prepared from IHP,
dodecasodium form and the corresponding free amine. IHP is first
protonated using a cation-exchange resin and is mixed to an
ethanolic solution of the desired amine. A first library of
IHP-ammonium salts was thus generated from commercially available
amines. The purpose was to assess the ability of amines to
transport IHP into a 1-octanol phase (K.sub.ow measurements) or
across erythrocyte membrane (P.sub.50 shift) as a function of the
amine structure. Therefore, we prepared IHP associated to primary,
secondary and tertiary amines bearing alkyl-, cycloalkyl- and
aromatic groups. In FIGS. 1-10 are represented the structures of
the amines and the ammonium salts of inositol hexaphosphate
constituting the present invention. FIGS. 1-7 depict monoamines and
monoammonium salts, and FIGS. 8-10 depict the polyammonium salts.
V. Partition Coefficients of IHP-ammoniums Derivatives
Partition coefficients relate to the distribution of a solute
between two immiscible liquid phases and are defined as the ratios
of concentrations (or molar fraction) of the distributed solute.
These data have been used to predict or rationalize numerous drug
properties such as quantitative structure/activity relationship (C.
Hansch, A. Leo Exploring QSAR; Fundamentals and Applications in
Chemistry and Biology, Washington D.C., 1995), lipophilicity (J.
Balzarini, M. Cools, E. D. Clercq Bioch. Biophys. Res Comm. 1986,
26, 6701-05) and pharmacokinetic characteristics. 1-Octanol has
been found to properly mimic biological membranes, and it has been
estimated than 1-octanol/water (K.sub.ow) partition coefficients of
more than 18 000 substances are now available in the literature (J.
Sangster Octanol/Water Partition Coefficients: Fundamentals and
Physical Chemistry, Chichester, 1997). ##EQU1##
Partition coefficients measurements. We synthesized a first series
of IHP derivatives, ionically associated to different aliphatic and
aromatic amines as well as amino-esters. Taking advantage of the 6
phosphate groups present in the IHP molecule, we measured, by
.sup.31 P-NMR, the partition coefficients of IHP salts between a
1-octanol phase and two different aqueous phases: water and human
serum. In FIG. 11 are represented the structures of the different
amines preassociated to IHP and the corresponding partition
coefficients K.sub.ow and K.sub.os. These coefficients are measured
after equilibration, at a concentration of 30 mM, close to the
typical concentration employed for the biological evaluations (the
standard concentration used for O.sub.2 -dissociation curve
measurements is 22 mM).
Choice of the aqueous phases. Because of the plethora of comparable
data described in the literature we first measured 1-octanol/water
partition coefficients K.sub.ow. To be even closer to physiological
conditions, we pushed this investigation further by measuring
1-octanol/human serum partition coefficients K.sub.os.
Interestingly, we observed that K.sub.os values are systematically
lower (by a factor of 2 to 5) than K.sub.ow values. Therefore, we
only measured K.sub.os 's when K.sub.ow values were significant. To
illustrate this observation we compared the partitions of 2 IHP
derivatives, SV75 and SV131, in different aqueous systems: water,
artificial serum (for its composition, see the Experimental
section) without and with albumin, and human serum. Table 1
summarizes these results.
TABLE 1 Partition coefficients (at 18 mM) of SV75 and SV131 with
different aqueous phases. K.sub.oa K.sub.oas K.sub.ow (artificial
serum (artificial serum K.sub.os (water) without albumin) with
albumin) (human serum) ##STR15## 0.411 0.088 0.041 0.031 ##STR16##
8.98 2.16 1.81 1.85
This simple series of results emphasizes the problems that can be
encountered when experiments are lead from water to real
physiological conditions. These results show the role that proteins
present in serum, such as albumin, can play: they suggest that
albumin is able to bind lipophilic amines, thus preventing IHP
uptake into the octanolic phase. This conclusion is in accordance
with the results published by Rim et al., who described a
comparative study between octanol/water an octanol/buffer partition
coefficients and correlated them to diffusion across brain
microvessel endothelium (S. Rim, K. L. Audus, R. T. Borchardt Int.
J. Pharm. 1986, 3, 79-84). Furthermore, albumin has been shown to
participate to cholesterol transport in blood, thus demonstrating
its ability to bind lipophilic compounds.
The analysis of this first series of partition coefficients can
already lead to interesting conclusions. Cyclic aliphatic amines
seem to display the best characteristics with regard to both
lipophilicity and water solubility. Indeed, we observed a very
significant increase in the K.sub.ow values from
cyclopentyl-(<10.sup.-3) to cyclooctyl-ammoniums (9.98).
Furthermore, the IHP cyclooctylammonium salt is still reasonably
soluble in water (the aqueous solubility limit is between 25 and 30
mM), whereas the corresponding n-octylammonium IHP salt presents a
solubility limit below 1 mM (thus rendering impossible its K.sub.ow
measurement as well as its biological evaluation as a soluble
drug). Hydrophobic amino-acids, even esterified, do not possess
satisfactory transport properties into the octanol phase, and will
not be considered as suitable lead compounds for further studies
aiming to IHP delivery into red blood cells.
As a comparison, the octanol/buffer partition coefficient of AZT,
an orally available anti-HIV drug has been determined to be 1.26
(the buffer being 100 mM sodium phosphate, pH 7.0) (T. P.
Zimmerman, W. Mahony K. L. Prus J. Biol. Chem. 1987, 262, 5748-54).
The same authors have shown that AZT diffuses across cell membranes
independently of the nucleoside transport system.
##STR17## ##STR18## Octanol/buffer partition coefficient: 1.26
0.064
The comparison between AZT K.sub.ow value and the best values we
obtained suggests that, at a concentration of 30 mM, the
lipophilicity of IHP-cycloalkylammoniums (especially cycloheptyl-
and cyclooctylammoniums) should be sufficient to allow IHP to be
delivered inside the erythrocyte.
However, new questions arise from this first study: the importance
of the number of lipophilic amines associated to IHP and the
evolution of IHP distribution in apolar phases as a function of the
drug concentration.
K.sub.ow dependence on IHP starting concentration. To answer these
important questions, we measured K.sub.ow variations of the two
best IHP salts, SV75 (9 cycloheptylammonium) and SV131 (11
cyclooctylammonium). The resulting curves are depicted in FIG. 12.
In all the aqueous phases tested, the variation of IHP partitioning
as a function of the starting concentration is always the same: the
distribution of IHP salts in octanol increases with the
concentration. It should be noticed that even at a concentration of
10 mM, IHP-cyclooctylammonium is equally distributed in the serum
and the octanol phase.
K.sub.os variation as a function of cyclooctylammonium
concentration. FIG. 13 shows two important characteristics of the
IHP transport into an apolar phase: at a constant IHP concentration
(22 mM), 8 equivalents of cyclooctylammoniums are required to reach
a K.sub.os value equal to 1, corresponding to an identical
distribution between human serum and octanol. Secondly, this
experiment shows that cyclooctylammonium ions, initially present in
their hydrochloride form in the organic phase, are able to extract
IHP, exclusively present as a sodium salt in human serum at the
beginning of the experiment.
This remarkable behavior suggests that, even if they are
accumulated in a cell membrane, transport molecules based on
lipophilic amines could continue to extract polyphosphates .
From a therapeutic point of view, if one wishes to perform
intravenous injection of highly concentrated IHP ammoniums salts,
the biocompatibility of the transport molecule has to be seriously
considered. Indeed, each IHP molecule is associated at least to 7
ammoniums, which means that the blood concentration in
alkyl-ammonium will be at least seven times greater than IHP
concentration. Consequently, the toxicity of the transport molecule
becomes a major issue. Therefore, the next step of this work is the
synthesis of polyamines bearing cycloalkyl groups. Once complexed
to IHP, the ammoniums should reach a greater affinity for IHP ex
vivo, and would provide a better delivery system. VI. Biological
Evaluation of the First Library
In FIG. 14 the different IHP derivatives have been classified by
amine types, the number of cations per IHP molecule as well as the
reference number are indicated beside each amine drawings. The
P.sub.50 shift values have to be compared to the control experiment
(underlined), corresponding to a test performed in the same
conditions but with IHP, sodium form. P.sub.50 values have been
measured in Professor Nicolau's laboratory in Boston (bioassay
conditions: 75 .mu.l of whole blood was incubated 2 minutes with
300 .mu.l of a 50 mM solution of IHP derivatives. The system was
then washed and 20 .mu.l were used for measurement of the
Hb-O.sub.2 dissociation curve at 37.degree. C.). See also FIGS.
15-17 for tables of P.sub.50 values at various osmolarities of
whole blood, and free hemoglobin that has been pre-incubated with
various ammonium salts of inositol hexaphosphate.
From the biological evaluation described above, several conclusions
can be drawn: the best results were obtained with two
IHP-cycloalkylamines SV81 (undeca cyclohexyl-ammonium) and SV75
(nona cycloheptyl-ammonium), at 220 mOsM, these IHP salts triggered
a 50% shift of the P.sub.50 value (compared to the control
experiment); aromatic (except tyrosine and phenylalanine
derivatives) and acyclic aliphatic amines (with a chain length
superior to 6 carbon atoms) decrease IHP solubility in aqueous
media to such an extent that their biological activity could not be
evaluated; amino-esters did not display transport properties; and
tributyl-ammonium salts and isoleucine-tBu ester provoked a fast
hemolysis of red blood cells, acting as strong detergents.
It is interesting to notice that good correlations between the
biophysical study (1-octanol/serum partition coefficients) and the
biological evaluation (P.sub.50 shift measurements), since both
techniques lead to the conclusion that cycloalkylamines displayed
the best properties for both lipophilicity (transport across the
erythrocyte membrane) and water solubility (which is an important
parameter for the bioavailibility of a potential drug). VII.
Polyamine Synthesis
The biological tests performed on whole blood as well as the
1-octanol/water partition measurements showed that amines bearing
cycloalkyl groups display the best properties in view of IHP
delivery into red blood cells. Multiple interactions between a
poly-anionic drug and a single transport molecule will increase the
binding strength between the two partners, thus preventing the
exchange, under physiological conditions with other cations present
in high concentration in serum. For this reason, lipophilic
polyamines seem to be the best candidates for transporting
polyphosphates, such as IHP or DPG, across non-polar biological
membranes. Thus, we optimized two general synthetic procedures in
order to obtain a new series of polyamines bearing cycloalkyl
groups: a reductive animation procedure leading to acyclic
tetra-amines, and a coupled acylation/borane-reduction procedure
for the preparation of macrocyclic polyamines.
The first optimized procedure lies on a single-step strategy, based
on a reductive animation between primary amines and different
cyclic ketones (Scheme 1). ##STR19## ##STR20##
This procedure proved efficient, giving good to excellent yields,
and versatile enough to allow reactions with hindered ketones such
as 2-decalone. The two central tetra-amine cores employed are TREN
(tris-(2-aminoethy)-amine) and BAP
(N,N'-bis-(3-aminopropyl)-piperazine). BAP is a linear tetra-amine
presenting two primary amines susceptible to be alkylated, whereas
TREN is a "ramified" molecule giving three possible N-alkylation
sites through reductive amination. Consequently, seven new
polyamines were obtained from these two central cores, either with
cyclohexyl groups (compounds SV98 and SV103), cycloheptyl group
(SV127), cyclooctyl-(SV147 and SV129), or 2-decalinyl groups (SV104
and SV111).
We then prepared a series of lipophilic polyamines derived from
CYCLAM, a well-studied and commercially available 14-membered ring
tetra-aza macrocycle. Macrocycles were chosen to strengthen the
interactions between polyphosphates and the poly-amine. To
illustrate this idea, FIG. 18 shows how CYCLAM, in a cis- or a
trans-decaline conformation may tightly bind 1,2-syn- or
1,2-trans-diphosphates, respectively.
One of the best procedures to obtain tetra-N-alkyl-cyclam
derivatives consists in a per-acylation of the four secondary
amines of cyclam, followed by reduction of the resulting
tetra-amide using an excess of borane-THF complex (Scheme 2).
##STR21##
Thus, the cyclam derivatives SV198 and SV224 were generated. Taking
into account the lack of solubility of IHP n-octylammonium salts in
water, we chose to introduce shorter chains, n-hexyl lipophilic
groups, for compound SV198. Compound SV224, with four
methyl-cyclohexyl groups, was designed to prevent water solubility
problems, and to insure a good partitionning into the apolar
phase.
Partition coefficient of IHP associated with polyamines. A second
library composed of di-, tri- and tetra-amines has been synthesized
using standard procedures and complexed to IHP. The structures are
listed in FIG. 19. Then, the partition coefficients K.sub.ow and
K.sub.os were measured by .sup.31 P-NMR. Table 2 tabulates P.sub.50
values measured with whole blood in the presence of the
IHP/polyammonium salts.
TABLE 2 P.sub.50 values measured with whole blood in the presence
of IHP/polyammonium salts. pH/37.degree. C. P50 Measure Osmolality
P50 HemoxAnalyzer of NAME CONTROL P50 Hemox+ Effector RATIOS
EFFECTOR mmHg mmHg EFF:washed (220-240) * (EFF) WB EFF:WB RBC mOsM
EFF:WB SV 89 fHb 16 41.7 7.2-7.3 Insoluble 0.25 umoles EFF SV 92
fHb 16 45.0 0.25 umoles EFF WB 41.4 58.5 7.2-7.3 84 1:0.375 WB 40.5
38.7 7.2-7.3 336 1:1.5 SV 94 fHb 16 41.0 7.2-7.3 Insoluble 0.25
umoles EFF SV 95 Insufficient Amount SV 97 fHb 16 46.7 7.2-7.3 0.25
umoles EFF WB 41.5 49.7 7.2-7.3 111 1:0.375 WB 40.5 40.0 7.2-7.3
343 1:1.5 SV102 fH 16 41.0 7.2-7.3 0.25 umoles EFF WB 41.5 75.0
7.2-7.3 82 1:0.375 WB 40.5 39.5 7.2-7.3 321 1:1.5 SV106/108 WB
Standard 26 Wash (-C) WB 29.5 33.4 7.2-7.3 151 1:0.375 WB 29.5 26.3
7.2-7.3 345 1:0.375 SV137 WB 29.5 32.0 7.2-7.3 268 1:0.375
Insufficient amount SV141 fHb 10.5 22.0 7.1-7.2 1: 1(2.5 mM:2.5 mM)
WB Standard 23.5 7.1-7.2 1:1.5 Wash (-C) WB Lyses no sufficient
1:0.375 RBC pellet WB 25 25.0 7.1-7.2 221 1:1.5 fHb = Free
Hemoglobin WB = Whole Blood
Once again, the poly-cycloalkylamines displayed better properties
than aromatic or acycic aliphatic ones. The tris-cycloheptyl-TREN
associated to IHP (SV137) showed the best partitioning
characteristics with K.sub.ow =2.70 and K.sub.os =0.64.
Disappointingly, a polyethyleneimine bearing cyclohexyl groups on
each primary amines once complexed to IHP gave a totally insoluble
product in both aqueous and organic solutions. At first sight, TREN
derivatives seem to be the best candidates for improving IHP
transport. The biological evaluation of this new library is still
in progress.
Biophysical and biological properties of DPG-Polyamine salts. In
order to compare the biophysical and the transport properties of
polyamines associated to another polyphosphate other than IHP, we
prepared poly-ammonium salts of the natural hemoglobin effector,
2,3-diphospho-glycerate (DPG). We selected, from the 2 IHP
libraries, the best amines and polyamines and prepared the
corresponding DPG salts. The structures of the DPG salts are
depicted in FIG. 20 along with their 1-octanol/water and
1-octanol/serum partition coefficients.
The different salts were then tested for P.sub.50 shift
measurements on whole blood. The results of these biological tests
are summarized in Table 3.
TABLE 3 Whole blood P.sub.50 measurements in the presence of
various DPG salts. pH/37.degree. C. Osmolality P50 P50 Measured of
NAME CONTROL P50 HemoxAnalyzer Effector RATIOS NAME EFFECTOR mmHg
mmHg Hemox+ (220-240) * EFFECTOR (EFF) WB EFF:WB EFF:washed RBC
mOsM EFF:WB (EFF) SV172 SV172 WB 36 Lyses 7.2-7.3 109 1:0.375 WB WB
36 36.0 7.2-7.3 311 1:0.375 WB SV164 SV164 WB 36 46.8 7.2-7.3 126
1:0.375 WB WB 36 36.0 7.2-7.3 337 1:0.375 WB SV174 SV174 WB 36 46.8
7.2-7.3 85 1:0.375 WB WB 36 36.0 7.2-7.3 289 1:0.375 WB WB 36 36.0
7.2-7.3 85 1:1.5 WB SV168 Insoluble SV168 SV216 Insoluble SV216
The results obtained with DPG salts are in good agreement with
those discussed above for the IHP ammonium salts. The
cyclooctylammonium salt SV164 displayed the best properties with
regard to partitioning in an octanolic phase and transport across
the red blood cell membrane. Once again, acyclic aliphatic
(poly)amines gave insoluble polyphosphaste salts in aqueous media.
VI. Methods of the Invention
In certain embodiments, the method of the present invention
comprises the step of administering to a subject red blood cells or
whole blood that has previously been treated ex vivo with a
compound or composition of the present invention, and wherein said
red blood cells or whole blood has been subsequently suitably
purified such that when said red blood cells or whole blood is
administered to a subject it is nontoxic to said subject.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject red blood cells or
whole blood that has previously been treated ex vivo with a
compound or composition of the present invention, wherein said red
blood cells or whole blood has been subsequently suitably purified
such that when said red blood cells or whole blood is administered
to a subject it is nontoxic to said subject, and wherein said
administration is intravenous.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject experiencing
ischemia red blood cells or whole blood that has previously been
treated ex vivo with a compound or composition of the present
invention, and wherein said red blood cells or whole blood has been
subsequently suitably purified such that when said red blood cells
or whole blood is administered to a subject it is nontoxic to said
subject.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject experiencing
ischemia red blood cells or whole blood that has previously been
treated ex vivo with a compound or composition of the present
invention, wherein said red blood cells or whole blood has been
subsequently suitably purified such that when said red blood cells
or whole blood is administered to a subject it is nontoxic to said
subject, and wherein said administration is intravenous.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject experiencing
cardiac arrhythmia red blood cells or whole blood that has
previously been treated ex vivo with a compound or composition of
the present invention, and wherein said red blood cells or whole
blood has been subsequently suitably purified such that when said
red blood cells or whole blood is administered to a subject it is
nontoxic to said subject.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject experiencing
cardiac arrhythmia red blood cells or whole blood that has
previously been treated ex vivo with a compound or composition of
the present invention, wherein said red blood cells or whole blood
has been subsequently suitably purified such that when said red
blood cells or whole blood is administered to a subject it is
nontoxic to said subject, and wherein said administration is
intravenous.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject experiencing a
heart attack red blood cells or whole blood that has previously
been treated ex vivo with a compound or composition of the present
invention, and wherein said red blood cells or whole blood has been
subsequently suitably purified such that when said red blood cells
or whole blood is administered to a subject it is nontoxic to said
subject.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject experiencing a
heart attack red blood cells or whole blood that has previously
been treated ex vivo with a compound or composition of the present
invention, wherein said red blood cells or whole blood has been
subsequently suitably purified such that when said red blood cells
or whole blood is administered to a subject it is nontoxic to said
subject, and wherein said administration is intravenous.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject experiencing a
stroke red blood cells or whole blood that has previously been
treated ex vivo with a compound or composition of the present
invention, and wherein said red blood cells or whole blood has been
subsequently suitably purified such that when said red blood cells
or whole blood is administered to a subject it is nontoxic to said
subject.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject experiencing a
stroke red blood cells or whole blood that has previously been
treated ex vivo with a compound or composition of the present
invention, wherein said red blood cells or whole blood has been
subsequently suitably purified such that when said red blood cells
or whole blood is administered to a subject it is nontoxic to said
subject, and wherein said administration is intravenous.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject experiencing
hypoxia red blood cells or whole blood that has previously been
treated ex vivo with a compound or composition of the present
invention, and wherein said red blood cells or whole blood has been
subsequently suitably purified such that when said red blood cells
or whole blood is administered to a subject it is nontoxic to said
subject.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject experiencing
hypoxia red blood cells or whole blood that has previously been
treated ex vivo with a compound or composition of the present
invention, wherein said red blood cells or whole blood has been
subsequently suitably purified such that when said red blood cells
or whole blood is administered to a subject it is nontoxic to said
subject, and wherein said administration is intravenous.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject afflicted with
sickle cell anemia red blood cells or whole blood that has
previously been treated ex vivo with a compound or composition of
the present invention, and wherein said red blood cells or whole
blood has been subsequently suitably purified such that when said
red blood cells or whole blood is administered to a subject it is
nontoxic to said subject.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject afflicted with
sickle cell anemia red blood cells or whole blood that has
previously been treated ex vivo with a compound or composition of
the present invention, wherein said red blood cells or whole blood
has been subsequently suitably purified such that when said red
blood cells or whole blood is administered to a subject it is
nontoxic to said subject, and wherein said administration is
intravenous.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject suffering from
hypotension red blood cells or whole blood that has previously been
treated ex vivo with a compound or composition of the present
invention, and wherein the red blood cells or whole blood has been
subsequently suitably purified such that when said red blood cells
or whole blood is administered to a subject it is nontoxic to said
subject.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject suffering from
hypotension red blood cells or whole blood that has previously been
treated ex vivo with a compound or composition of the present
invention, wherein said red blood cells or whole blood has been
subsequently suitably purified such that when said red blood cells
or whole blood is administered to a subject it is nontoxic to said
subject, and wherein said administration is intravenous.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject suffering from
arteriosclerosis red blood cells or whole blood that has previously
been treated ex vivo with a compound or composition of the present
invention, and wherein said red blood cells or whole blood has been
subsequently suitably purified such that when said red blood cells
or whole blood is administered to a subject it is nontoxic to said
subject.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject suffering from
arteriosclerosis red blood cells or whole blood that has previously
been treated ex vivo with a compound or composition of the present
invention, wherein said red blood cells or whole blood has been
subsequently suitably purified such that when said red blood cells
or whole blood is administered to a subject it is nontoxic to said
subject, and wherein said administration is intravenous.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject suffering from
altitude sickness red blood cells or whole blood that has
previously been treated ex vivo with a compound or composition of
the present invention, and wherein said red blood cells or whole
blood has been subsequently suitably purified such that when said
red blood cells or whole blood is administered to a subject it is
nontoxic to said subject.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject suffering from
altitude sickness red blood cells or whole blood that has
previously been treated ex vivo with a compound or composition of
the present invention, wherein said red blood cells or whole blood
has been subsequently suitably purified such that when said red
blood cells or whole blood is administered to a subject it is
nontoxic to said subject, and wherein said administration is
intravenous.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject suffering from
diabetes red blood cells or whole blood that has previously been
treated ex vivo with a compound or composition of the present
invention, and wherein said red blood cells or whole blood has been
subsequently suitably purified such that when said red blood cells
or whole blood is administered to a subject it is nontoxic to said
subject.
In certain embodiments, the method of the present invention
comprises the step of administering to a subject suffering from
diabetes red blood cells or whole blood that has previously been
treated ex vivo with a compound or composition of the present
invention, wherein said red blood cells or whole blood has been
subsequently suitably purified such that when said red blood cells
or whole blood is administered to a subject it is nontoxic to said
subject, and wherein said administration is intravenous.
In certain embodiments, the method of the present invention
comprises the step of adding to mammalian blood a compound or
composition of the present invention.
In certain embodiments, the method of the present invention
comprises the step of adding to plasma comprising mammalian
erythrocytes a compound or composition of the present invention.
IX. Pharmaceutical Compositions
In another aspect, the present invention provides pharmaceutically
acceptable compositions which comprise a therapeutically-effective
amount of one or more of the compounds described above, formulated
together with one or more pharmaceutically acceptable carriers
(additives) and/or diluents. A natural requirement for any
pharmaceutically acceptable composition is that it comprise a
nontoxic compound of the present invention. We are aware that many
of the modem drugs of great benefit have started out as toxic
substances. Ongoing research in our laboratories is directed
towards nontoxic compounds of ammonium salts and anionic allosteric
effectors. The pharmaceutical compositions of the present invention
may be specially formulated for administration in solid or liquid
form, including those adapted for the following: (1) oral
administration, for example, drenches (aqueous or non-aqueous
solutions or suspensions), tablets, boluses, powders, granules,
pastes for application to the tongue; (2) parenteral
administration, for example, by subcutaneous, intramuscular or
intravenous injection as, for example, a sterile solution or
suspension; (3) topical application, for example, as a cream,
ointment or spray applied to the skin; or (4) intravaginally or
intrarectally, for example, as a pessary, cream or foam.
The phrase "therapeutically-effective amount" as used herein means
that amount of a compound, material, or composition comprising a
compound of the present invention which is effective for producing
some desired therapeutic effect in at least a sub-population of
cells in an animal at a reasonable benefit/risk ratio applicable to
any medical treatment.
The phrase "pharmaceutically acceptable" is employed herein to
refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals A without excessive toxicity, irritation, allergic
response, or other problem or complication, commensurate with a
reasonable benefit/risk ratio.
The phrase "pharmaceutically-acceptable carrier" as used herein
means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material, involved in carrying or
transporting the subject compound from one organ, or portion of the
body, to another organ, or portion of the body. Each carrier must
be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations. X. Administration of the Compounds of the Present
Invention
In another aspect, the current invention provides methods of
administering to a subject pharmaceutical compositions comprised of
a nontoxic ammonium salt of an anionic allosteric effector. Many
techniques currently exist for delivering drugs or other
medicaments to body tissue. These include, among possible others,
oral administration, injection directly into body tissue such as
through an intramuscular injection or the like, topical or
transcutaneous administration where the drug is passively absorbed,
or caused to pass, into or across the skin or other surface tissue
and intravenous administration which involves introducing a
selected drug directly into the blood stream. Techniques and
formulations generally may be found in Remmington's Pharmaceutical
Sciences, Meade Publishing Co., Easton, Pa.
Exemplification
The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention, and are not intended to
limit the invention.
Physical Chemistry
Human serum was purchased at the "Centre de Transfusion Sanguine de
Strasbourg". Artificial serum was based on blood composition :
[Na.sup.+ ]=140 mM, [Mg.sup.2+ ]=1 mM, [K.sup.+ ]=5 mM, [Ca.sup.2+
]=1 mM, [Cl.sup.- ]=106 mM, [PO.sub.4.sup.3- ]=1 mM,
[SO.sub.4.sup.2- ]=0.5 mM, [CO.sub.3.sup.2- ]=30 mM, [Br.sup.-
]=0.5 mM, bovine serum albumin (fraction V, >98%) 30 g.l.sup.-1,
pH=7.41.
Partition coefficients. IHP derivatives were dissolved in 1 ml of
aqueous phase at a concentration of 30 mM in an eppendorf. 1 ml of
1-octanol was then added and each sample was shaken at 36 rpm
during 12 hours. The equilibrated biphasic solutions were
centrifuged for 2 hours at 3000 rpm. 350 .mu.l of the octanolic
phase was first taken off with a syringe and put directly in the
NMR tube. 350 .mu.l of the aqueous phase was then taken off.
.sup.31 P-NMR spectra were performed on a Bruker AC-300 apparatus.
An external standard (triphenylphosphine oxide, 60 mM in d.sub.6
-DMSO) was placed inside the NMR tubes to allow both the locking
process of the apparatus and an accurate integration of IHP peaks.
Partition coefficients were determined as the ratio of IHP
integrations, relative to the external standard, in the octanol and
aqueous phases. The detection limits of this technique do not allow
partition coefficients measurements below 10.sup.-3.
General Procedures
IHP polyammoniums Synthesis. A 100 mM IHP dodecasodium salt
solution was applied on a cation exchange column (dowex WX8-200,
H.sup.+ form) and eluted with distilled water. The fractions
containing the perprotonated IHP were collected and poured onto an
ethanolic solution of the desired amine. The solution was then
concentrated in vacuo, redissolved in ethanol or in a 1/1
toluene/EtOH mixture and reconcentrated. The IHP-ammonium salts
were characterized by .sup.1 H- and .sup.31 P-NMR.
IHP-, DPG-ammonium salts Syntheses
SV44 Inositol hexaphosphate, hepta 3-hydroxy-quinuclidinium
salt
The general procedure was applied from commercial IHP, dodecasodium
form, and 3-hydroxy-quinuclidine. White solid.
.sup.1 H-NMR (200 MHz, D.sub.2 O) .delta.4.89 (d, J=10.4 Hz, 1H),
4.42 (q, J=10.4 Hz, 2H), 4.17 (m, 10H), 3.62 (m, 7H), 3.25 (m,
35H), 2.18 (m, 35H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.1.88 (1P), 1.26 (2P),
0.67 (2P), 0.25 (1P).
SV46 Inositol hexaphosphate, nona piperidinium salt
The general procedure was applied from commercial IBP, dodecasodium
form, and piperidine. White solid.
.sup.1 H-NMR (200 MHz, D.sub.2 O) .delta.4.25 (q, J=9.9 Hz, 2H),
3.98 (m, 3H), 3.01 (m, 36H), 1.58 (m, 54H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.2.55 (1P), 1.59 (2P),
1.34 (2P), 1.17 (1P).
SV48 Inositol hexaphosphate, nona phenylalanine-methyl-ester
The general procedure was applied from commercial IHP, dodecasodium
form, and phenylalanine methyl-ester. White solid.
.sup.1 H-NMR (200 MHz, D.sub.2 O) .delta.7.31 (m, 45H), 4.88 (d,
J=10.0 Hz, 1H), 4.41 (q, J=10.0 Hz, 2H), 4.21 (m, 12H), 3.71 (m,
27H), 1.17 (bd, J=10.0 Hz, 18H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.2.18 (1P), 1.51 (2P),
1.00 (2P), 0.59 (1P).
SV51 Inositol hexaphosphate, hexa dihydro-quinolidinium salt
The general procedure was applied from commercial IHP, dodecasodium
form, and dihydro-quinolidine. White solid.
.sup.1 H-NMR (200 MHz, D.sub.2 O) .delta.7.51 (m, 24H), 4.83 (m,
7H), 4.48 (q, J=9.8 Hz, 2H), 4.21 (m, 3H), 3.01 (m, 12H), 2.55 (m,
6H), 2.13 (m, 6H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.2.18 (1P), 1.51 (2P),
0.92 (2P), 0.54 (1P).
SV52 Inositol hexaphosphate, hepta 2-norbornyl-ammonium salt
The general procedure was applied from commercial IHP, dodecasodium
form, and 2-norbornylaminer. White solid.
.sup.1 H-NMR (200 MHz, D.sub.2 O) .delta.4.85 (d, J=9.9 Hz,1H),
4.36 (q, J=9.9 Hz, 2H), 4.08 (m, 3H), 3.48 (m, 7H), 2.31 (m, 14H),
1.97 (bt, J=11.0 Hz,1H), 1.28 (m, 49H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.2.67 (1P), 1.84 (2P),
1.46 (2P), 1.34 (1P).
SV53 Inositol hexaphosphate, nona decahydroquinolinium salt
The general procedure was applied from commercial IHP, dodecasodium
form, and decahydro-quinoline. White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.4.31 (q, J=9.7 Hz, 2H),
3.4-2.8 (m, 27H), 1.9-1.0 (m, 117H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.2.84 (1P), 2.01 (3P),
1.59 (2P).
SV55 Inositol hexaphosphate, hepta phenylalanine-ethyl-ester
The general procedure was applied from commercial IHP, dodecasodium
form, and phenylalanine ethyl-ester. White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.7.22 (m, 35H), 4.33 (q,
J=9.6 Hz, 2H), 4.21 (m, 24H), 3.07 (m, 7H), 1.07 (t, J=7.1 Hz, 21
H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.2.63 (1P), 1.63 (5P).
SV57 Inositol hexaphosphate, octa isoleucine-tbuthyl-ester
The general procedure was applied from commercial IHP, dodecasodium
form, and isoleucine tBu-ester. White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.4.35 (q, J=9.7 Hz, 2H),
4.09 (m, 3H), 3.84 (d, J=4.0 Hz, 8H), 1.91 (bs, 8H), 1.5-0.8 (m,
120H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.1.88 (1P), 1.30 (2P),
0.75 (2P), 0.25 (1P).
SV58 Inositol hexaphosphate, dodeca diisopropylammonium salt
The general procedure was applied from commercial IHP, dodecasodium
form, and diisopropylamine. White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.4.33 (q, J=9.9 Hz, 2H),
4.05 (m, 3H), 3.38 (h, J=4.6 Hz, 12H), 1.18 (d, J=4.6 Hz,
120H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.2.09 (1P), 1.38 (2P),
0.75 (2P), 0.33 (1P).
SV59 Inositol hexaphosphate, octa proline-t-buthyl-ester
The general procedure was applied from commercial IHP, dodecasodium
form, and proline tBu-ester. White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.4.35 (m, 10H), 4.02 (m,
3H), 3.29 (m, 16H), 2.26 (m, 8H), 1.95 (m, 24H),1.36 (m, 72H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.2.21 (1P), 1.42 (2P),
0.92 (2P), 0.54 (1P).
SV68 Inositol hexaphosphate, hepta tyrosine-ethyl-ester salt
The general procedure for IHP ammonium salt preparation was applied
from IHP dodecasodium form and the commercially available
amine.
.sup.1 H NMR (300 MHz; D.sub.2 O) .delta.7.06 (d, J=8.04 Hz, 20H),
6.77 (d, J=7.70 Hz, 20H), 4.37 (q, J=9.8 Hz, 2H), 4.41 (m, 33H),
4.22 (m, 7H), 3.96 (m, 14H), 3.44 (bs, 14H), 1.04 (t, J=7.3 Hz, 6
11H).
.sup.31 P NMR (121 MHz; D.sub.2 O) .delta.3.00 (1P), 2.75 (2P),
2.58 (2P), 1.50 (1P).
SV74 Inositol hexaphosphate, undeca-adamantylammonium salt
The general procedure for IHP ammonium salt preparation was applied
from IHP dodecasodium form and the commercially available
amine.
.sup.1 H NMR (300 MHz; CD.sub.3 OD/CDCl.sub.3 3/1) .delta.4.53 (q,
J=9.87 Hz, 2H), 4.14 (m, 3H), 3.41 (bs, 11H), 1.9-0.9 (m,
154H);
.sup.31 P NMR (121 MHz; CD.sub.3 OD/CDCl.sub.3 3/1) .delta.2.72
(1P), 2.30 (2P), 1.84 (3P).
SV75 Inositol hexaphosphate, nona-cycloheptylammonium salt
The general procedure for IHP ammonium salt preparation was applied
from IHP dodecasodium form and the commercially available
amine.
.sup.1 H NMR (300 MHz; D.sub.2 O) .delta.4.34 (q, J=9.95 Hz, 2H),
4.01 (m, 3H), 3.26 (qi, J=5.13 Hz, 9H), 1.95 (m, 18H), 1.70-1.35
(m, 90H);
.sup.31 P NMR (121 MHz; D.sub.2 O) .delta.3.22, 2.76, 2.47,
2.09.
SV78 Inositol hexaphosphate, undeca-cyclopentylammonium salt
The general procedure for IHP ammonium salt preparation was applied
from IHP dodecasodium form and the commercially available
amine.
.sup.1 H NMR (300 MHz; D.sub.2 O) .delta.4.29 (q, J=9.30 Hz, 2H),
3.98 (m, 3H), 3.53 (bs, 11H), 1.9-0.9 (m, 88H);
.sup.31 P NMR (121 MHz; D.sub.2 O) .delta.2.72 (1P), 2.30 (2P),
1.84 (3P).
SV81 Inositol hexaphosphate, undeca-cyclohexylammonium salt
The general procedure for IHP ammonium salt preparation was applied
from IHP dodecasodium form and the commercially available
amine.
.sup.1 H NMR (300 MHz; D.sub.2 O) .delta.4.26 (q, J=9.60 Hz, 2H),
3.94 (m, 3H), 2.91 (bs, 11H), 1.9-0.9 (m, 110H);
.sup.31 P NMR (121 MHz; D.sub.2 O) .delta.3.18, 2.68, 2.39,
2.09.
SV92 Inositol hexaphosphate, penta
(.+-.)-(trans)-1,2-cyclohexyldiammonium salt
The general procedure for IHP ammonium salt preparation was applied
from IHP dodecasodium form and the commercially available
amine.
.sup.1 H NMR (300 MHz; D.sub.2 O) .delta.4.81 (m, 1H), 4.31 (q,
J=9.40 Hz, 2H), 4.09 (m, 3H), 3.57 (m, 2.5H), 3.22 (m, 5H), 2.93
(m, 2.5H), 2.08 (bd, 5 H), 1.80-1.25 (m, 30H);
.sup.31 P NMR (121 MHz; D.sub.2 O) .delta.3.43 (2P), 2.39 (1P),
2.09 (2P), 1.97 (1P).
SV94 Inositol hexaphosphate, penta
cyclohexyl-(1,3-bismethylammonium) salt
The general procedure was applied from commercial IHP, dodecasodium
form, and 1,3-bis-aminomethyl-cyclohexane (mixture of isomers).
White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.4.37 (m, 3H), 4.18 (bd,
0.5H), 2.82 (m, 13H), 2.66 (m, 5H), 1.93 (m, 5H), 1.8-1.1 (m, 32H),
0.89 (q, J=12.2 Hz, 7H), 0.66 (q, J=12.2 Hz, 3H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.5.69 (2P), 4.32 (3P),
3.56 (1P).
SV97 Inositol hexaphosphate, nona N-cyclohexyl-piperidinium
salt
The general procedure was applied from commercial IHP, dodecasodium
form, and N-cyclohexyl-piperidine. White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.4.91 (d, J=10.1 Hz, 1H),
4.35 (q, J=10.0 Hz, 2H), 4.09 (bq, J=9.3 Hz, 3H), 3.20 (s, 36H),
3.02 (s, 36H), 2.67 (m, 9H), 1.93 (m, 18H), 1.59 (bd, 9H), 1.3-0.9
(m, 45H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.2.63 (1P), 2.26 (2P),
2.05 (1P), 1.76 (1P).
SV99 Inositol hexaphosphate, hexa N,N-dimethyl-cyclohexylammonium
salt
The general procedure was applied from commercial IHP, dodecasodium
form, and N,N-dimethyl-cyclohexylamine. White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.4.84 (bd, 1H), 4.41 (q,
J=9.9 Hz, 2H), 4.14 (m, 3H), 3.11 (t, J=11.4 Hz, 6H), 2.80 (s,
36H), 1.95 (bd, 12H), 1.81 (bd, 12H), 1.58 (bd, 6H), 1.3-1.0 (m,
36H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.1.55 (1P), 1.09 (2P),
0.63 (2P), 0.13 (1P).
SV72 Inositol hexaphosphate,
penta-(N,N'-dibenzyl)-ethylenediammonium salt
The general procedure for IHP ammonium salt preparation was applied
from IHP dodecasodium form and the commercially available
amine.
.sup.1 H NMR (200 MHz; CD.sub.3 OD/CDCl.sub.3 3/1) .delta.7.45-7.21
(m, 80H), 4.99 (d, J=10.0 Hz, 1H), 4.79 (s, 40H), 4.44-4.21 (m,
5H), 3.91 (bs, 32H), 2.98 (bs, 32H);
.sup.31 P NMR (121 MHz; CD.sub.3 OD/CDCl.sub.3 3/1) .delta.3.00
(1P), 2.75 (2P), 2.58 (2P), 1.50 (1P).
SV89 Inositol hexaphosphate, octa menthyl-1,8-diammonium salt
The general procedure was applied from commercial IHP, dodecasodium
form, and 1,8-diamino-menthane. White solid.
.sup.1 H-NMR (200 MHz, D.sub.2 O) .delta.4.45 (q, J=9.7 Hz, 2H),
3.97 (m, 3H), 1.9-0.8 (m, 150H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.4.52 (2P), 2.80 (1P),
2.68 (1P), 2.51 (2P).
SV94 Inositol hexaphosphate, penta
cyclohexyl-(1,3-bismethylammonium) salt
The general procedure was applied from commercial IHP, dodecasodium
form, and 1,3-bis-aminomethyl-cyclohexane (mixture of isomers).
White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.4.37 (m, 3H), 4.18 (bd,
0.5H), 2.82 (m, 13H), 2.66 (m, 5H), 1.93 (m, 5H), 1.8-1.1 (m, 32H),
0.89 (q, J=12.2 Hz, 7H), 0.66 (q, J=12.2 Hz, 3H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.5.69 (2P), 4.32 (3P),
3.56 (1P).
SV95 Inositol hexaphosphate, penta
(.+-.)-(1,2-trans-diphenyl)-ethylenediammonium salt
The general procedure was applied from commercial IHP, dodecasodium
form, and 1,2-trans-diphenyl-ethylenediamine. White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.7.25 (m, 50H), 5.03 (bd,
1H), 4.81 (s, partially overlapped with the D.sub.2 O peak), 4.49
(q, J=10.0 Hz, 2H), 4.25 (m, 3H).
SV97 Inositol hexaphosphate, nona N-cyclohexyl-piperidinium
salt
The general procedure was applied from commercial IHP, dodecasodium
form, and N-cyclohexyl-piperidine. White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.4.91 (d, J=10.1 Hz, 1H),
4.35 (q, J=10.0 Hz, 2H), 4.09 (bq, J=9.3 Hz, 3H), 3.20 (s, 36H),
3.02 (s, 36H), 2.67 (m, 9H), 1.93 (m, 18H), 1.59 (bd, 9H), 1.3-0.9
(m, 45H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.2.63 (1P), 2.26 (2P),
2.05 (1P), 1.76 (1P).
SV101 Inositol hexaphosphate, bis (N.sup.1,N.sup.3
-cyclohexyl)-dipropylenetriammonium salt
The general procedure was applied from commercial IHP, dodecasodium
form, and triamine SV91. White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.4.90 (bm, 1H), 4.35 (bm,
2H), 4.15 (bm, 3H), 3.06 (t, 16H), 2.07 (m, 16H), 1.77 (bs, 8H),
1.59 (bs, 4H), 1.3-1.0 (m, 24H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.2.18 (1P), 1.38 (2P),
0.88 (3P).
SV102 Inositol hexaphosphate, tris
tri-(N-cyclohexyl-2-amino-ethyl)-ammonium salt
The general procedure was applied from commercial IHP, dodecasodium
form, and tetramine SV98. White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.4.81 (bd, J=9.9 Hz, 1H),
4.34 (q, J=9.9 Hz, 2H), 4.01 (m, 3H), 3.1-2.7 (m, 45H), 1.99 (m,
15H), 1.65-1.5 (m, 30H), 1.4-1.0 (m, 45H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.2.43 (1P), 2.30 (1P),
2.13 (2P), 1.80 (2P).
SV106 Inositol hexaphosphate, tetra
N,N'-di-(3-(N-cyclohexyl-amino)-propyl)-piperazinium salt
The general procedure was applied from commercial IHP, dodecasodium
form, and tetramine SV103. White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.4.90 (bd, J=9.8 Hz, 1H),
4.34 (q, J=9.8 Hz, 2H), 4.03 (m, 3H), 2.98 (bt, J=7.1 Hz, 24H),
2.60 (bs, 24H), 2.50 (bt, J=7.1 Hz, 24H), 1.99 (bs, 16H), 1.80 (m,
32H), 1.60 (m, 8H), 1.40-1.05 (m, 44H).
SV137 Inositol hexaphosphate, tris
tri-(N-cycloheptyl-2-amino-ethyl)-ammonium salt
The general procedure was applied from commercial IHP, dodecasodium
form, and tetramine SV127. White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.4.81 (bd, J=9.3 Hz, 1H),
4.39 (q, J=9.3 Hz, 2H), 4.12 (m, 3H), 3.25 (m, 6H), 3.22 (m, 10H),
2.94 (m, 10H), 2.02 (m, 12H), 1.70-1.40 (m, 60H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.2.36 (1P), 1.23 (2P),
0.67 (2P), 0.44 (1P).
SV141 Inositol hexaphosphate, tri
N,N'-di-(3-(N-cyclooctyl-amino)-propyl)-piperazinium salt
The general procedure was applied from commercial IHP, dodecasodium
form, and tetramine SV129. White solid.
.sup.1 H-NMR (300 MHz, D.sub.2 O) .delta.4.89 (bd, J=10.4 Hz, 1H),
4.30 (q, J=9.7 Hz, 2H), 4.03 (m, 3H), 3.25 (m, 8H), 2.99 (bt, J=7.1
Hz, 14H), 2.65 (bs, 16H), 2.45 (bt, J=7.1 Hz, 14H), 1.96 (m, 28H),
1.80-1.20 (m, 84H).
.sup.31 P-NMR (121 MHz, D.sub.2 O) .delta.7.87 (1P), 7.72 (2P),
7.13 (1P), 6.60 (2P).
SV202 Inositol hexaphosphate, bis N,N',N",N'"-tetrahexyl-cyclam
salt
The general procedure was applied from commercial IHP, dodecasodium
form, and tetramine SV198. White solid.
.sup.1 H-NMR (300 MHz, CD.sub.3 OD/CDCl.sub.3 1/1) .delta.4.96 (bd,
J=11.1 Hz, 11H), 4.25 (t, J=9.3 Hz, 1H), 4.15 (t, J=9.3 Hz, 2H),
3.4-2.6 (bm, 44H), 2.17 (s, 4H), 1.54 (m, 12H), 1.29 (m,60H), 0.86
(t, J=6.0 Hz, 24H).
.sup.31 P-NMR (121 MHz, CD.sub.3 OD/CDCl.sub.3 1/1) .delta.1.69
(2P), 0.93 (1P), 0.51 (2P), 0.25 (1P).
EXAMPLE 1
This example shows that ammonium salts of Inositol Hexaphosphate
(IHP) improve the dissociation of oxygen from hemoglobin following
incubation with whole blood. A. Effectors
See FIGS. 1-3. B. Blood Preparations
Whole blood was collected from one subject. The blood was stored in
a Vacutainer with Solution A (ACD) and stored at 4-8.degree. C.
To isolate red blood cells, whole blood (3 mL) was placed on top of
test tube containing 9 mL of Histopaque 1119 (Sigma Diagnostics
Inc.) and 1 mL of Saline buffer. Following centrifugation the
supernatant and buffy coat were removed and the pellet containing
RBCs were washed three times in 10 mL HBS. C. Buffers
HBS=HEPES Buffered Saline,
HBS was used as the standard buffer for experiments. HBS 7.42
(r.t.) was ideal to keep pH of experiments at 7.28-7.32 (37.degree.
C.). 20 mM HEPES 130 mM Sodium Chloride
HEPES, (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid])
C.sub.8 H.sub.18 N.sub.2 O.sub.4 S F.W.=238.3 pK.sub.a =7.5 pH:
6.8-8.2 CAS# 7365-45-9
HBS+ HBS 20 .mu.L Bovine Serum Albumin (BSA) per 5 mL HBS (TCS
Medical Products Company) 15 .mu.L Antifoaming per 5 mL HBS (TCS
Medical Products Company) pH: 7.2-7.4 Osmolarity: 290-320 mOsM
HBS.cndot.BSA mL HBS Plus 20 .mu.L BSA
saline, 0.9% Sodium Chlorida, Injection USP Each 100 mL
contains:
900 mg NaCl 154 mEq/L Sodium 154 mEq/L Chloride pH: 5.0 Osmolarity:
308 mOsM
BIS-TRIS buffered saline,
(bis[2-Hydroxyethyl]minotris[hydroxymethyl]methane), (Sigma). 20 mM
Bis-Tris 140 mM Sodium Chloride pH: 7.45 Osmolarity: 294 mOsM D.
Procedures
Preparation of Effector Stock: Effector stock was prepared at
100-120 mM (Molal solution) using water or Bis-Tris Buffer.
Effector characteristics prior to incubation were:
Concentration: 30 mM Osmolarity: 170-340 mOsM pH: 7.1-7.4 (at
37.degree. C.)
Incubation: Whole blood (75-300 .mu.L) was incubated with 200 .mu.L
of effector at 37.degree. C. for 5-10 min. (see Summary of Results
below).
Washes: After incubation of whole blood with/without effector,
blood cells were washed four times with Saline buffer or HBS (BSA)
by centrifugal pelleting to remove exogenous effector and to
evaluate hemolysis. After final centrifugation, pellet was not
resuspended.
Blood Oxygen Dissociation Reading: Blood Oxygen Dissociation of
samples were determined using a Hemox Analizer Model B (TCS Medical
Products Company, New Hope, Pa.). The sample chamger contained:
Control: 2.5-3.0 mL of HBS+ 25 .mu.L Whole blood Effector
evaluation: 2.5-3.0 mL of HBS+ 10-20 .mu.L Pelleted Blood Cells
incubated with Effector
All readings were made at 36.7-37.2.degree. C. and at pH 7.28-7.32.
The P.sub.50 s were calculated from the Dissociation Curves
compared to same day control P.sub.50.
Example 1
Summary of Results
P.sub.50 of Whole Blood Pre-Incubated with Effector (Low
Osmolarity) (All incubations and measurements at 37 +/- 0.2 C.)
P.sub.50 P.sub.50 CONTROL EFF:WB CONC. CONC OSMOL. Volume WB mmH
EFF EFF:WB EFF pH pH Ratio EFFECTOR mmHg g mM mM mOsM EFF. EFF:WB
EFF:WB ICP6 27.5 39 30 22 220 7.23 1:0.375 NH4-IHP 26 54 30 22 106
7.29 1:0.375 28.5 43 30 22 106 7.28 1:0.375 30 53.5 30 22 94 7.43
1:0.375 SV73 38 57 30 22 68 7.54 1:0.375 WB = whole blood; EFF =
allosteric effector; ICP6 = nona cyclohexylammonium tri sodium
inositol hexaphosphate; SV73, see FIG. 3. The control value for
whole blood's P.sub.50 varies due to aging of the blood. Aging is
accompanied by the degradation of natural allosteric effectors by
native phosphotases.
See also FIGS. 4-9. E. Conclusion
Ammonium Salts of IHP increase the P.sub.50 of whole blood in
comparison to the sodium salts of these two allosteric effectors at
osmolarities less than 280 mOsM.
Incorporation by Reference
All of the patents and publications cited herein are hereby
incorporated by reference.
Equivalents
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. Such
equivalents are encompassed by the following claims.
* * * * *